HELICOBACTER PYLORI INFECTION IN EGYPTIAN PATIENTS WITH DYSPEPSIA: DIAGNOSTIC, DEMOGRAPHIC, ENDOSCOPIC AND CLINICAL CHARACTERISTICS.

Background and Study Aims: Helicobacter pylori (H. pylori ) is considered as a public health problem, especially in developing countries. Understanding the epidemiological aspects of H. pylori infection is important and helpful in clarifying the consequences and complications of infection. The aim of this study was to assess the prevalence of H. pylori infection in dyspeptic patients and to study the demographic, assess the prevalence of H. pylori infection in dyspeptic patients and to study the demographic, clinical and endoscopic characteristics of H. pylori infected patients. We tried to evaluate the role of combined testing assays for H. pylori diagnosis. stool antigen results by plotting the optical density on the y-axis against the concentration of the calibrator values on the x-axis. Negative results: < 15 ng/ml while positive results: > 20 ng/ml.

Background and Study Aims: Helicobacter pylori (H. pylori) is considered as a public health problem, especially in developing countries. Understanding the epidemiological aspects of H. pylori infection is important and helpful in clarifying the consequences and complications of infection. The aim of this study was to assess the prevalence of H. pylori infection in dyspeptic patients and to study the demographic, endoscopic and clinical characteristics of H. pylori infected patients. Patients and Methods: A total of 113 adult patients with dyspepsia were enrolled in this study. They underwent upper gastrointestinal endoscopy for obtaining four antral biopsies, patients were considered to be infected with H. pylori when they had positive results of rapid urease and/or H.pylori stool antigen tests and confirmed by detection of H. pylori 16S rRNA gene in the extracted DNA from gastric biopsy specimens by Polymerase Chain Reaction (PCR) assay. Results: Sixty (53.1%) dyspeptic patients (17-76 years old) were confirmed to be infected with H. pylori. Age, sex, smoking history and taking spicy food had no significant correlation to the acquisition of H. pylori infection. Considering the PCR assay on gastric biopsy specimens as the gold standard, excellent agreement was found with both rapid urease and H. pylori stool antigen tests. Rapid urease test (91.7%) was more sensitive than H. pylori stool antigen test (83.3%), while both tests have specificity of 100%. Upon endoscopy; gastritis was revealed in 27(45%) and 10

DNA extraction and detection of H. pylori 16S rRNA gene by conventional PCR assay:
Genomic DNA was extracted from gastric biopsy specimens using QIAamp DNA min kit (Qiagen, USA) according to manufacturer guidelines. PCR assay was performed in a volume of 50ul with approximately 5 μg of extracted DNA, 200 μM (each) dNTPs, 25 pmol for each primer (Table 1), 1.5 μM Magnesium Chloride and 1unit of Taq polymerase (Gotaq Flexi DNA, M8305, Promega, Inc, USA) in PCR buffer. The reaction was done in PTC-100 TM thermal cycler (MJ Research, USA), programmed as follows: denaturing at 95°C for 5 min, followed by 37 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1 min, and final extension at 72°C for 5 minutes. PCR product was separated on 2% agarose gel with ethidium bromide, and 50bp ladder used as DNA molecular weight standard. In PCR assay, a negative control (lacking DNA) was included. PCR products were analyzed under UV light. Size of expected amplicon was 110bp [15].
Statistical analysis: Data were described in terms of frequencies (number of cases) and relative frequencies (percentages). P-value < 0.05 was considered as statistically significant. All statistical calculations were done using computer programs Microsoft Excel 2007 (Microsoft Corporation, NY, U.S.A.).

Diagnostic performance of different assays used for diagnosis of H. pylori infection:
Regarding results of different assays used for diagnosis of H. pylori infection among 113 dyspeptic patients included in the study; for conventional PCR assay, 60 (53.1%) patients showed positive H. pylori DNA in gastric biopsy specimens, 55(48.6%) gastric biopsy specimens had positive rapid urease test and 50 (44.2%) patients had positive H. pylori stool antigen. The diagnostic performance analysis of different assays on gastric biopsies and stool specimens, considering PCR assay on gastric biopsy specimens as the gold standard, was; 60 (53.1%) patients were diagnosed to be infected with H. pylori by positive results of rapid urease and /or H. pylori stool antigen tests and confirmation by detection of 16S rRNA in gastric biopsy specimens. The sensitivity rate of rapid urease test (91.7%) was higher than that of H. pylori stool antigen test (83.3%) and for specificity rate of both tests was 100%. Excellent agreement was found with rapid urease test and stool antigen test, with Kappa coefficient of 0.912 and 0.824; respectively ( Figure 1 and Table 2) Chisholm et al [15] 229  , and 85% (51/60) patients were from urban region while 15% (9/60) were from rural region (P value=0.97). Half of the H. pylori infected patients (31/60, 51.7%) were above 51years, 46.7% (28/60) were between 21 and 50 years, while only one patient (1.7%) was below 20 years (P value > 0.5). H. pylori infection increases with age, and was possibly found more in population above 51 years.

Risk factors associated with H. pylori infected patients:
The risk factors associated with H. pylori infection include smoking and eating spicy food (13/60; 21.7%; each) and both factors were detected in 10 % (6/60) of patients (P value > 0.5).   [23]. In contrast, studies which reflect good hygienic conditions and less crowded environments reported a much lower prevalence from several European countries [16,24].
In the current study, 85% of H. pylori infected patients were from urban region where these patients are living in a low-income urban region with low educational level. Similarly, previous studies reported higher prevalence rates of H. pylori infection in low-income urban areas [10]. Jaka et al [24] also reported significantly higher prevalence rates of H. pylori infections in rural areas compared to urban areas.
In the present study, although the prevalence of H. pylori infection was higher in males, however, no statistically sex difference was found as it has also been found in other studies conducted in developing countries, there were no gender differences in the risk of acquisition of infection [18]. On the contrary Niknam, et al [25], reported predominant H. pylori positivity in females.
Age was shown to have an effect on the prevalence of H. pylori infection with lower rates in subjects younger than 20 years old [26]. Ford and Axon [27] reported that H. pylori infection prevalence increases with age between 7% and 87%. Among adults, the H. pylori infection rate increases with age [24].The current study the prevalence of H. pylori infection increases with age; however the differences were not significant. Hu et al [28] reported that the highest prevalence of H. pylori was in 40-49 years age group, they explained that these subjects always tend to dine out which increases the chance of exposure to H. pylori infection.
Smoking is a social stigma that is fast growing into an epidemic. It has been elucidated in molecular studies that smoking causes the expression of cagA gene in the gastric milieu. This gene could play a role in the transformation of an ulcerated gastric lining into a malignant tumor [29]. In the current study, smoking was associated with H. pylori infection in 21.7%. Such result was contradictory to Bakka and Salih [26] study and was consistent with previous study by Hu et al [28] who reported that the use of tobacco had been shown to impair the immune system, therefore contributing to an increased chance of H.pylori infection.

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H. pylori infection could also be related to eating habits [30]. In the current study, eating spicy food, as a risk factor, is associated for H. pylori infection. These results were in agreement with Hu et al [29] who reported that spicy food and pickle food are risk factors for H. pylori infection. Spicy food and pickle food may decrease the ability of gastric mucosa to prevent H. pylori infection and a high intra-gastric salt content may destroy the mucosal barrier, leading to inflammation and damage such as diffuse erosion and degeneration in the stomach.
Dyspepsia is a common gastrointestinal disorder and could present as dysmotility or ulcerative like dyspepsia. It is considered as the most common indication for gastric upper endoscopy [31]. The current study showed that 56.7 % of the H.pylori infected patients were presented with dyspepsia. Such results were lower than those found by Vilaichone et al [32] from Thailand. In the Western World, the prevalence of dyspepsia was ranged between 25 to 50% [25]. Savarino et al [33] documented that up to 70% of reflux patients have typical reflux symptoms (i.e. heartburn and/or regurgitation) in the absence of endoscopic visible oesophageal mucosal injuries.
More than 50% of the world's population is infected with H. pylori [27], however, 10% only will develop peptic ulcer disease and1%-2% will develop gastric malignancy [34]. The upper gastrointestinal endoscopy of the studied H. pylori infected patients revealed that, the most common findings were gastritis (45%) followed by peptic ulcer (16.7%). Our results were in agreement with Ngoyi et al [35] and lower than Rasheed et al [36]. Feliciano et al [37] reported that during upper endoscopy, patient's mucosa was distributed into non-peptic ulcer dyspepsia in 64.7% and peptic ulcer in 35.3%of cases. Other endoscopic findings (31.7%) were observed among the studied H. pylori infected patients. Moschos et al [38] showed that H. pylori eradication may positively influence GERD symptoms. Ngoyi et al [35] had detected hiatal hernia in 1.6% of the examined cases. Another study from Egypt by Safwat et al [39] showed that there is significant association between H. pylori infection and the occurrence and the severity of portal hypertensive gastropathy (which may be associated with esophageal varices) in patients with HCV-related liver cirrhosis, thus, eradication of H.pylori may be beneficial to improve portal hypertensive gastropathy.
Of the several available diagnostic tests for detection of H. pylori infection, the choice depends on the sensitivity, specificity, reproducibility, availability cost, and rapidity of the results. There is a need for a reference method to be used as "gold standard" to detect patients truly infected. Unfortunately, none of the currently used methods is able to further this criterion [40]. Nowadays, studies show that the PCR assay may be slightly superior to other diagnostic methods for detection of H. pylori from different clinical samples and to verify the bacterium eradication after treatment. The need for a limited amount of bacteria enables PCR to recognize infection when other tests are negative due to low bacterial density [41].
In the current study, 53.1% of the studied patients were H. pylori positive by rapid urease test and/or stool antigen test and confirmed by detection of 16S rRNA gene by PCR assay in panel of gastric biopsy specimens. We tried to combine the results of two techniques, and compare with results of each method being evaluated. Using PCR assay as gold standard, the diagnostic performance of the studied assays revealed that both invasive biopsy-based rapid urease test and H. pylori stool antigen test as a non-invasive method had excellent agreement.
Concerning first-choice diagnostic tests for H. pylori, the American College of Gastroenterology recommended biopsy-based tests. Among these, rapid urease test is the most popular test in clinical practice but has high variable number of false-negative results according to a meta-analysis [42]. False positive results can occur if other urease containing organisms are present in sufficient quantity. The sensitivity of urease test is also affected by the amount of bacteria in the biopsy [43]. In the current study, sensitivity and specificity rates of rapid urease test was 91.7% and 100% respectively detection of H. pylori infection from the biopsy site and the pooled 3 biopsies sites helps to detect even more patients with H. pylori infection. Our results were comparable with Parihar et al [44] and higher than Allahverdiyev et al [45]. Nevoa et al [46] found that the rate in the detection of H. pylori by the molecular method was significantly higher when compared to the rapid urease test.
Several non-invasive tests have been developed to diagnose H. pylori. H.pylori stool antigen has been detected successfully, using purified antibodies by immunoassay. It is a reliable method to diagnose an active infection and to evaluate the eradication of H.pylori infection. However, if the concentration of H.pylori antigen becomes low, false negativity may also be reported [47]. In the current study H. pylori stool antigen test showed a sensitivity rate of 83.3% and specificity rate of 100%. Higher sensitivity rates (92.4%) and a specificity rate of 100% was reported by Okuda et al [48]. Comparable results were recorded by Me´graud et al [49]. A lower sensitivity rate and a comparable specificity rate were revealed by Korkmaz et al [50]. Saha et al [51] found that H.pylori 232 stool antigen test was superior to upper gastrointestinal endoscopy for detection of H.pylori infection and recommended for initial testing for H.pylori infection in dyspeptic patients before initiating treatment and before carrying out any invasive procedure such as endoscopy.
In conclusion, H. pylori infection rate in Egyptian patients with dyspepsia was high and gastritis was the most revealed finding upon endoscopy. No risk factors were associated with H. pylori infection among the studied adult patients. Combined rapid urease and stool antigen tests can be relied upon for detecting H. pylori infection. Noninvasive method could be used for H. pylori re-testing when invasive tests are negative or could not be done.

Conflict of interest:-
We have no conflict of interest to declare.