ISOLATION AND DETECTION OF HELICOBACTER PYLORI FROM PATIENTS SUFFERING FROM PEPTIC ULCER USING BIOCHEMICAL TESTS AND MOLECULAR TECHNIQUES

Helicobacter pylori is the causative agent of most cases of gastritis. There is no established gold standard for the diagnosis of H. pylori infection. A reliable diagnosis is crucial to conf irm that eradication therapy has been successful. Eighty gas tric biopsy and blood samples were obtained from fasting Jordanian patients with Esophago-Gastro-Dou denoscopy (EGD). Several diagnosis tests for H. pylori infections were used and compared including: Cultu re, microscopic examination, histopathology, Rapid Urease Test (RUT), serology, biochemical tests, antibiotic susceptibility test a nd molecular method. Forty two patients were considere H. pylori positive in both histopathology examination and RUT test. On the other hand, 57 pat ient were detected to have anti-IgA, IgG H. pylori antibody positive by ELISA test. Ten patients had e quivocal results but not in both tests. A total of 19 biopsy samples were positive for H. pylori according to culture test. This result was confirm ed by endoscopic examination, urease, catalase and oxidas e. A high percentages of resistance to vancomycin, polymyxin B and amoxicillin was observed (100, 100 and 94.7%, respectively) with various degree of sensitivity to all of the first line of antibiotics . Molecular technique (PCR) was used to detect CagA gene which appeared positive in 14 patients. We con clude that the histopathology and RUT tests are reliable invasive diagnosis for H. pylori. However, culture test appear to be the most impor tant (if the therapy failed) to detect antibiotic susceptibility to H. pylori strains.


INTRODUCTION
The discovery of Helicobacter pylori in 1982 was the starting point of a revolution concerning the concepts and management of gastroduodenal diseases (Megraud and Lehours, 2007). Helicobacter pylori, originally classified as Campylobacter pylori (Al-Sulami et al., 2010). Selfingestion experiments and experiments with volunteers demonstrated that these bacteria can colonize the human stomach, thereby inducing inflammation of the gastric mucosa (Morris et al., 1991). These initial data stimulated for further study, which showed that gastric colonization with H. pylori can lead to upper gastrointestinal disorders, such as chronic gastritis, peptic ulcer disease, gastric mucosaassociated lymphoid tissue lymphoma and gastric cancer. The severity of the inflammation is likely to underlie H. pylori-related diseases (Abdalsadeg et al., 2012). Although most individuals do not develop adverse clinical consequences from H. pylori colonization, between 10 and 15% will experience some form of symptomatology Science Publications AJBB and the clinical result of the infection will be determined by complex interactions between host and bacterial factors. While the host factors remain unknown, the identification of specific bacterial factors is continuously advancing (Cogo et al., 2011).
H. pylori is a gram negative bacterium typically appearing as a curved rod or short spiral. It is believed to be one of the more common pathogenic infections of man, with prevalence rates reaching 30-60% in developed countries, depending on age and socioeconomic status (Vinette et al., 2002). The mode of transmission and other aspects of the epidemiology of H. pylori infection still remain unclear (Tiwari et al., 2005). Several virulence factors that aid H. pylori in colonization of the host and contribute to disease development have been identified by a variety of methods. One of these factors is the urease enzyme that H. pylori needs to survive in the low-pH gastric lumen as it makes its way to the gastric mucosa, which has a more neutral pH (Montecucco and Rappuoli, 2001). H. pylori also requires several motility and chemotaxis genes for colonization, presumably so that it can locate and move to its preferred site of infection and remain there (Ottemann and Lowenthal, 2002).
Diagnosing H. pylori infection is sometimes difficult (Tiwari et al., 2005). Accurate diagnosis is essential for the effective treatment and management of infections caused by this organism (Abdalsadeg et al., 2012). H. pylori infection can be diagnosed by invasive techniques (endoscopy with biopsies for histology, culture and a rapid urease test) (Vaira and Vakil, 2001). Culture and identifying H. pylori in gastric biopsy require experience and dexterity, as identification and culturing are sometimes difficult. Microscopy and rapid urease test can be highly specific if strictly performed, but they are based on biopsy specimens and thus are theoretically prone to sampling error, as in the case of culture (Yoshida et al., 1998). Since invasive methods are expensive, less invasive methods such as serological examination of blood and the urea breath test have become more popular (Zagari et al., 1999). However, positive results by blood serology do not necessarily allow delineation of active H. pylori infection. Urea breath tests require expensive specialized equipment and reagents and sometimes become apparently positive in culture negative patients (Bazzoli et al., 1997).
Molecular methods like Polymerase Chain Reaction (PCR) have been used extensively for the diagnosis of H. pylori from gastric biopsy specimens, saliva, faeces and archival specimens, as well as for detecting clarithromycin resistance (Zsikla et al., 2006). PCR yields information on the presence of potential virulence markers in the strain, which might have implications for the development of severe disease or efficacy of eradication (Ricci et al., 2007). Different primers have been utilized and some have been developed into commercial kits. Different loci have been used as the target for the amplification: 16S rRNA; A-B-and Curease; flaA; CagA; vacA and heat-shock protein (hsp). Real-time results can be obtained using light-cycle technology (Ricci et al., 2007).
The present study was therefore carried out to test some feasible non-invasive and invasive methods for the rapid diagnosis of H. pylori among infected patients suffering from various gastric maladies using biochemical tests and molecular techniques.

Biopsy Samples
A total of 80 biopsy patients (30 males and 50 females) aged from 18-81 years were collected from two different local hospitals. The patients were suffering from dyspepsia and advised by the doctors to have clinical indications for an endoscopy in the gastrointestinal clinic of the hospitals. All the patients with dyspepsia undergoing endoscopy were asked to fill the inclusion questioner. Biopsies of gastric tissue were collected from the corpus or the antrum or corpus and antrum of the patient's stomach. Three biopsies were taken from each patient. Then, specimens were sent for histopathologic study, gram staining, sensitivity and rapid urease test.
The taken biopsies were classified according to the performed test. The biopsy used for histopathology examination was transported to the histopathology laboratory with 10% buffered formalin for at least 24 h. The biopsy used for culture was transported to the laboratory with 2-2.5 mL −1 Tryptical Soy Broth (TSB) as a transport medium. The biopsy which used for RUT was tested immediately at the department of gastroenterology and the result examined within 1 h.

Blood Samples
Blood samples (3-5 mL −1 ) were taken before each patient's enter to endoscopic unit and before initiation of any therapy. The blood was kept at room temperature for 1 h; the clot was removed by centrifuging at 1,000-2,000xg for 10 min in the centrifuge. The resulting supernatant was designated serum. Following centrifugation, the samples were immediately transferred into a clean, sterile eppendorf tube using a Pasteur pipette. The samples were maintained at 2-8°C while handling. Then the serum was stored at-20°C until using for Enzyme-Linked Immunosorbent Assay (ELISA) test.

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Samples that are hemolyzed, icteric or lipemic were excluded from the study.

Histopathologic Examination
This test was performed by the laboratory technical in the hospital and read by the doctor following standard procedure (Dixon et al., 1996). The gastric biopsies were fixed in 10% buffered formalin for at least 24 h and then embedded in paraffin. In each case, three sections of the tissues were cut at 0.3 micron, de-paraffinized and hydrated in descending grades of alcohol, cut in sequential 4-µm sections. One section was stained with routine haematoxylin and eosin stain. The second section was stained with modified Giemsa stain to demonstrate the presence of H. pylori. The H. pylori were identified as curved rods on the luminal surface of the gastric epithelial cells. The third section was stained with alcian blue/Periodic Acid Schiff's stain to demonstrate the presence of intestinal metaplasia.

Culture of H. Pylori from Biopsy Sample
The antral biopsy specimens were transported to the microbiology laboratory immediately within less than one hour in 2-2.5 mL tryptic soy broth as a transport medium. The biopsy specimens were first ground in a sterile mortar with the aid of a sterile fine glass rod until the formation of homogenate. The biopsies were inoculated at Columbia Agar (CA) plates supplemented with 8% sheep blood, vancomycin (10 mg mL −1 ), trimethoprim lactate (5 mg L −1 ), amphoteracin-B (5 mg mL −1 ), polymixin-B (2500 units mL −1 ) and incubated in a microaerobic atmosphere (10 CO2, 85 N2, 5% O2) (Sigma, Aldrich) at 37°C for 5-7 days. The bacteria were identified as H. pylori by Gram staining, colony morphology, positive oxidase, catalase and urease reactions (Balows et al., 1991). Bacteria were sub-cultured using the same conditions into two different media: Columbia agar with supplements and brain heart infusion broth.

Rapid Urease Test
Biopsy specimens were tested for urease activity by using the "HelicotecUT®Plus" test.

Urease Test
The urease test was applied according to MacFaddin (2000). The pure isolates were inoculated heavily on the entire surface of urea agar and stab with loop wire. The tubes were inoculated at 37°C in the incubator. The formation of purple color was examined after 4 h.

Oxidase Test
The isolates were tested for oxidase activity by using oxidase test strip.

Catalase Test
The isolates were tested for catalase activity by using slide (drop) method in which the sterile wire loop is stacked on the surface of the pure colony and transferred to a microscope slide and one or two drop of 3% H2O2 were added onto the organism on the slide and observed for immediate oxygen bubble formation.

DNA Extraction from Biopsy Samples
Specimens of gastric biopsy were collected in a sterile container containing digestion buffer (100 mM NaCl, 10 mM Tris-HCl (pH 8.0) 0.5% SDS) and frozen at-70°C until using. Genomic DNA was isolated from all samples by the Cetyltrimethyl Ammonium Bromide (CTAB) method with an additional initial digestion with lysozyme buffer and incubation with the buffer at 37°C for 24 h, then according to the standard protocol. Extensive care was taken to avoid contamination during all steps of collection and preparation of the samples.

PCR Amplification
Two oligonucleotide primers designated CagA-F (5'-AATACACCAACGCCTCCAAG-3') and CagA-R (5'-TTGTTGCCGCTTGCTCTC-3') was used. The amplified product of these two primers with DNAs was a 395 bp fragment. The template DNA [2 µL −1 ] was added to 18 µL −1 of the reaction mixture containing 1X PCR buffer [50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5% (v/v) Triton X-100], 1.5 mM MgCl2, 200 uM concentrations of each dNTPs, 10 pmol of each primer and 1U of Taq polymerase. PCR amplification was performed according to the following program: Initial denaturation at 96°C for 5 min 40 cycles with each cycle consisting of 94°C for 1 min, 62°C for 1 min and 72°C for 2 min. The final cycle included a 10 min extension step to ensure full extension of the PCR products. In each batch of PCR assays the negative control consisted of all the reagents of the master mix except the template DNA. The PCR-amplified products were analyzed by agarose gel electrophoresis. The gel was stained with ethidium bromide and examined under UV transilluminator for the presence of the amplified DNA.

Statistical Analysis
Standard method was used to calculate sensitivity and specificity for the result of diagnostic test. Sensitivity relates to the test's ability to identify positive results. The sensitivity of a test is the proportion of people that are known to have the disease who test positive for it. This can also be written as:

Data Collection Analysis
A total of 240 biopsy samples were collected from 80 Jordanian patients. The objectives of this study were described for the patients and they agreed to be in the population of the study. They filled the required case history data which included civilian information about the patients (sex, age,), the symptoms they suffering from (epigastric pain, nausea, vomiting….) and their medical history (other diseases and medications).
The results of the case history data indicated that there were 38.75% (31 out of 80) males and 61.25% (49 out of 80) females with age range 18-81 years and mean age 42.55 years. 96.25% (77 out of 80) of the patients were not on any medications while 3.75% (3 out of 80) used aspirin, famodar, NAISD before presentation to the outpatients, but not in a regular way. Forty four patients were suffering from epigastric pain for 6-12 months and 67 suffering from heartburn and 55 suffering from the symptoms after eating.

Serum Enzyme-Linked Immunosorbent Assay Test (ELISA)
The 80 patients consented to be randomly tested against H. pylori serum antibody (IgG and IgA). Anti-H. pylori IgG antibodies were present in 56 (70%), while anti-H. pylori IgA antibodies present in 47(58.75%) ( Table 1). The test depends on the optical density for the formed colored because of interaction between antibodies (if they present in serum) with the substrate solution form yellow color. The results of the absorbance and concentrations of Anti-H. pylori, IgG and IgA antibodies for the 80 samples are presented as standard curve in (Fig. 1).

Microscopic Examination, Oxidase, Urease and Catalase Tests
Nineteen Helicobacter pylori isolates were obtained from the whole 80 biopsy specimen samples which cultured from the transport media on Columbia agar supplemented with selective antibiotic and 8% sheep blood, in a microaeriphilic jar for 5-7 days. The isolations were identified firstly according to colony morphology that appeared transparent almost with poor growth. Secondly, they were microscopically examined after stained with Gram stain; the isolates were Gram negative, long and spiral shape. Biochemical examinations with catalase, urease and oxidase activity were applied to all of the 19 detected bacterial colonies that are positive for H. pylori ( Table 3). The results indicated that all of the 19 isolates were oxidase positive, transparent colonies and appeared spiral shaped under the microscopic examination.

Antibiotic Susceptibility Test
The 19 H. pylori positive isolates (according to the biochemical tests and gram stain) were tested for their susceptibility to 8 different types of antibiotics. The results indicated that the isolates were 100% VAncomycin (VA) and Polymyxins B (PB) resistant, 94, 84.2 and 47.4% were resistant to AmoXicillin (AX), Kanamycin (K), Claithromycin (CLA) respectively and 84.2, 89.5 and 94.7% were sensitive to TEtracycline (TE), TriMethoprim (TM) and RifAmpin (RA) respectively (Table 4).

Molecular Detection of H. pylori Isolates
To confirm the results of the conventional methods that mentioned above for diagnosis of H. pylori (ELISA, RUT test, histopathology, culture and biochemical tests), PCR technique was used. Genomic DNA was extracted from the 19 isolates (Fig. 3) and the concentration was measured by nanodrop instrument. The extracted genomic DNA was tested to detect the presence of CagA gene in the H. pylori isolates. The CagA gene was amplified with a specific primers (5'-AATACACCAACGCCTCCAAG-3') and (5'-TTGTTGCCGCTTGCTCTC-3'). The amplified genomic DNA of the isolates produced a single PCR band of about 395 bp in size (Fig. 4). The diagnosis test results and their histopathology examination for the 19 patients are presented in Table 5.

Statistical Analysis
Using the statistical standard statistical equation, the sensitivity and specificity values for the H. pylori diagnosis test were calculated as shown in Table 6. All the tests have a high sensitivity for detection of H. pylori except the culture test which produced about (47.5%) sensitivity values, where histopathology and culture tests shows 100% specificity.   19 (100)

DISSCUSION
The prevalence of Helicobacter pylori differs both between and within countries, with high rates of infection being associated with low socioeconomic status and high densities of living (Goodman and Cockburn, 2001;Hazel and Francis, 2002). Approximately, 40 and 80% of adult individuals in developed and developing countries are infected respectively (Timothy and Martin, 1995). However, the percentage of infected people increases with age, since 50% of infected people were those over the age of 60 compared with around 10% between 18 and 30 years (Brown, 2000). This was not the case in this study, since the highest percentage of patients was among young people ranging from 40-60 years. In a large French cross-sectional study, a significantly lower prevalence of H. pylori infection was observed in females as compared with males (Broutet et al., 2001). However, in this study a highest range of infection was found among females.
The results of this study indicated that 53.75% of symptomatic patients were infected with H. pylori. The infection was associated with variable gastrointestinal illness, chronic gastritis, intestinal metaplasia and ulceration disease. This was in agreement with Pilotto et al. (1998) who reported that chronic superficial gastritis associated with H. pylori infection is a significant predisposing factor for the development of peptic ulcer, atrophic gastritis, gastric lymphoma and gastric adinocarcinoma.
Many tests are available for diagnosis of H. pylori infection. Invasive tests, such as culture, histopathology and biopsy urease test that are required endoscopic biopsy of gastric tissue. Non-invasive tests, such as antibody that was detected in the serum also have been used. Patients with alarming symptoms should undergo endoscopy for the diagnosis of H. pylori infection. When endoscopy is clinically indicated, the test of first choice is the urease test on an antral-biopsy specimen. It permits cheap and rapid detection of urease activity in the biopsy material, with a sensitivity of 79 to 100% and a specificity of 92 to 100%. Sensitivity can be improved by additional biopsies, but false negative results are observed in patients with active or recent bleeding and in patients taking antibiotics or antisecretory compounds (Suerbaum and Michetti, 2002). If the urease test is negative, additional biopsy specimens stored in fixative can be sent for histological examination. Rapid urease test has been shown to be as reliable as histological examination. There were only minor differences in accuracy between the RUT and histopathology invasive tests for H. pylori infection in this population. RUT may be recommended as the first choice since a result is obtained within one hour (Suerbaum and Michetti, 2002).
The present study was aimed to compare the results of mostly used three invasive methods: Culture test, rapid urease test and histopathological stained based detection of H. pylori infection. The results of histopathology and RUT kit were almost similar, 43 (53.75%) patients were RUT positive and 40 (50%) were histology positive. The sensitivity values for these two tests were 100% to H. pylori, while the specificity value was found to be 93.5 for RUT and 100% for histopathology. The result of RUT is read after 60 min but the median positive reaction time was only 1 min. The results of this study were in agreement with (Ricci et al., 2007) who mentioned that the sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 98.2, 99, 99, 97.9 and 98.5%, respectively. The sensitivity and specificity of histology for the diagnosis of H. pylori varies from 53 to 90%, depending partly on the clinical setting, partly on the density of colonization and partly on the experience of the histopathologist.
H. pylori serologic test is cheap and widely used for the diagnosis of H. pylori infection in patients before treatment. Although approved laboratory-based tests have sensitivities and specificities of the commercial H. pylori, antibody tests seemed to vary between 60 and 100% (Salomaa et al., 2004). In this study, the result of serological test indicated that Anti-H. pylori IgG antibodies were present in 56 (70%) of the samples while 47(58.75%) have IgA anti-H. pylori antibodies. Specificity and sensitivity for IgG were 60 and 100 and were 80 and 100% for IgA, respectively. The results of this study indicated that the accuracy of the H. pylori IgG and IgA tests were in agreement with the previous reference. The accuracy of serological tests is strongly dependent on the prevalence of H. pylori infection. Although it has been recommended that antibody assays be evaluated locally, this has rarely been carried out for different age groups. To avoid any misjudgment in the validation of serological tests for H. pylori antibodies in adult subjects, the validation should be carried out Science Publications AJBB separately for different age groups with special emphasis not only on the known H. pylori status but also on the presence of atrophic gastritis (Salomaa et al., 2004).
Primary isolation of H. pylori from a biopsy specimen is a difficult process. Several factors, which are difficult to control, cause difficulty with the culturing of this bacterium. Another study showed that the percentage of culture positive specimen was 31.94% (Kagar et al., 2011). Patchy distribution of the organism on the gastric mucosa, contamination of biopsy forceps, presence of oropharangyeal flora, loss of viability of the organism during transportation, all these may be responsible for a poor negative predictive value associated with culture of H. pylori (Meunier et al., 1997). Culture of H. pylori with antibiotic-sensitivity testing is not routinely performed for the initial diagnosis of H. pylori infection, but it is recommended after the failure of second-line therapy (Bazzoli, 2001). Guidelines for performing antibioticsusceptibility tests of H. pylori have been developed by the National Committee for Clinical Laboratory Standards (Performance standards for antimicrobial susceptibility testing, 1999). In this study, the rate of isolation of H. pylori from the antral biopsy specimens were 47.5% and the sensitivity and specificity of this test were 47.5, 100% respectively. This is in agreement with Ricci et al. (2007) who indicated that culture test has a high specificity (100%) and the sensitivity is often lower.
Bacterial contamination of the medium was frequent. The contaminant bacteria were Pseudomonas spp., Proteus spp. and Klebsiella spp., the source of which could be contaminated biopsy forceps and contamination during obtaining, transporting and preparing of the defibrinated sheep blood added to the classic Columbia agar. The growth rate of H. pylori on this medium was slow, as 5 to 7 days were needed for the colonies to appear. Columbia blood agar is a nonselective medium used for many years alone or in combination with other non-selective and selective media for culturing H. pylori from antral biopsy specimens taken from peptic ulcer patients during upper gastrointestinal endoscopy (Fresnadillo Martinez et al., 1997). The isolation rate of H. pylori using this medium alone is very variable. Results as low as only 28.5% total isolation rate were reported by some authors (Piccolomini et al., 1997).
PCR has been used extensively for the diagnosis of H. pylori from gastric biopsy specimens, saliva, faeces and archival specimens, as well as for detecting clarithromycin resistance (Ricci et al., 2007). The targets of these PCR methods included urease A (ureA) gene cag A gene, phosphosamine mutase (glmM) gene and 23S rRNA gene (Tomatari et al., 2010). In this study, PCR technique was used to determine the virulence factor CagA in H. pylori; 73.6% of the biopsy specimens were CagA positive. Smith et al. (2011) and his colleagues detected H. pylori in 35% of their patients using a PCR method in Nigeria.
The disadvantages of PCR as a routine test are that it is a technically demanding and expensive test compared to culture, histology and the rapid urease tests. It requires special laboratory conditions with separate facilities for each stage of the technique and as it is highly sensitive, it is subject to false-positive results by contamination. A positive result detected by any of the molecular techniques does not only indicate current infection but will also detect the DNA or dead organisms (Lisby, 1999). Molecular biology is useful in the study of pathogenicity and immunization vis-à-vis Helicobacter pylori. The recent description of the genome of H. pylori causes molecular biology at the forefront in research on H. pylori. PCR is considered the most sensitive method for the detection of microorganisms. This would make the test particularly useful in the evaluation of eradication. Other indications include the detection of H. pylori outside the stomach, although H. pylori is present in small numbers among many other microorganisms causing false positive in other tests based on urease and culture (Hassina and Ahmed, 2013).
Antibiotic resistance has increasingly been recognized as a major cause of treatment failure for H. pylori infection. Primary antimicrobial resistance against clarithromycin and metronidazole is now commonplace in several countries (Poon et al., 2002). Regional variations in susceptibility and resistance patterns may be ascribed to differences in local antibiotic prescription practices, antibiotic usage in the community and mass eradication programs for H. pylori infection as part of gastric cancer prevention strategies. These factors may well be expected to influence success of eradication therapy (Wong et al., 2002). In this study, all the 19 isolated strains showed various degree of sensitivity to all the first line of antibiotics namely trimethoprim, rifampin, clarithromycin, kanamycin and tetracycline. The results indicated that the isolates were 100% vancomycin and polymyxins B resistant and 94%, amoxicillin resistant.

ACKNOWLEDGMENT
The researchers are expressing their appreciations to Al-Balqa` Applied University for the financial support of this study.