Sabaa F1.doc J Bagh College Dentistry Vol. 27(3), September 2015 Establishment of the Oral Diagnosis 85 Establishment of the possible association between the presence of Helicobacter pylori in the saliva and gastric biopsy by using polymerase chain reaction technique in association with oral manifestation of peptic ulcer disease Sabaa R. Thamer, B.D.S. (1) Sahar Al-Ani, B.D.S., M.Sc., Ph.D. (2) ABSTRACT Background: Helicobacter pylori are important gastrointestinal pathogen associated with gastritis, peptic ulcers, and an increased risk of gastric carcinoma. There are several popular methods for detection of H. pylori (invasive and non-invasive methods) each having its own advantages, disadvantages, and limitations, and by using PCR technique the ability to detect H. pylori in saliva samples offers a potential for an alternative test for detection of this microorganism. Materials and methods: The study sample consists of fifty participants of both genders, who undergo Oesophageo- gastrodudenoscopy at the Gastroenterology Department of Al-Kindy Teaching Hospital Baghdad/ Iraq, during five months period from January 2014 to May 2014. They were grouped into 32 participants with PUD (case group) and 18 healthy participants (control group). A full-mouth examination was performed for every patient; saliva and gastric samples from both groups were obtained. Helicobacter pylori were detected in gastric biopsies by histological examination by using H & E stain, and Polymerase Chain Reaction (PCR) was carried out on the oral samples. Results: Helicobacter pylori DNAwas determined by PCR in oral samples in 88% patients and in gastric biopsies by histology in 86% patients, and in both samples in 84% patients.It was highly significant to find simultaneous presence for those have H. pylori in stomach also have such microorganism in the mouth P < 0.05 and there was an excellent correlation between detecting H. pylori simultaneously in both stomach and mouth. If we screen for stomach H. pylori through detecting this microorganism in the mouth; saliva samples is highly sensitive (98%) but not very specific. Conclusion: Helicobacter pylori saliva test has high sensitivity, specificity, and accuracy for the diagnosis of H. pylori infection in Iraqi population. The test can be clinically applied as a routine diagnostic tool for H. pylori infection this could permit not only a target for therapeutic procedures but also a monitoring tool for the efficacy of therapy. It seems to overcome some limitations of the conventional invasive techniques. Key words: Helicobacter pylori - Oral infection - H. pylori saliva test – PUD. (J Bagh Coll Dentistry 2015; 27(3):85-88). INTRODUCTION Peptic ulcer is a breach in the gastric or duodenal mucosa down to the sub mucosa (1). In most cases the etiology of ulcer is unknown yet (2).The normal stomach mucosa maintains a balance between protective and aggressive factors. Some of the main aggressive factors are gastric acid, abnormal motility, pepsin, bile salts, use of alcohol and non-steroidal anti- inflammatory drugs (NSAID), as well as infection with microorganisms (Helicobacter pylori) (3). The association of H. pylori with peptic ulcer disease has been observed (4). This study has been shade light on the role of (H. pylori) as the commonest bacterial infection worldwide, it infects more than 50% of the worlds’ population (5), and as a main cause of PUD (6).Helicobacter pylori first reported in 1983 by Warren and Marshall, (7). H. pylori (initially termed Campylobacter pyloridis) is an important human pathogen (8). (1)Master Student, Department of Oral Diagnosis, College of Dentistry, Baghdad University. (2)Assistant professor, Department of Oral Diagnosis, College of Dentistry, Baghdad University. H.pylori is a Gram-negative spiral bacterium that may colonize the human gastric mucosa and establish a life-long infection (9). Colonization with H. pylori is not a disease by itself (10). It has been shown that infection certainly makes the occurrence of ulcers more likely, Infection linked with the development of chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma (11,12). Because of the importance of this bacterium in the development of PUD, Rasmussen (13) evaluated the association between the presence of H. pylori in gastric biopsies and in the saliva of the same individuals. The relationship between gastric symptoms and H. pylori DNA in saliva, however, is unclear. It could be possible that the oral cavity is the initial site of infection (14). Many diagnostic methods have been developed to detect H. pyloriincluding the urea breathtests, rapid urease tests, and measurement of anti-H. Pylori antibody from serum and urine, special histologic staining and immunostaining, and stool antigen testing. Many PCRmethods targeting putative H. pylori specific genes have also been reported (15,16).The goal of this study was todetermine the simultaneous presence of H. J Bagh College Dentistry Vol. 27(3), September 2015 Establishment of the Oral Diagnosis 86 pylori both in the oral cavity and gastric mucosa in patients suffering from PUD and to standardize a feasible non-invasive method for the rapid diagnosis of H. pylori in salivary secretion of infected patients suffering from PUD by using PCR analysis. MATERIALS AND METHODS The study sample consists of fifty patients of both genders with age range (15-65) years; who undergo Oesophageogastrodudenoscopy during five months period from January 2014 to May 2014. They were grouped into 32 participants with PUD (case group) and 18 healthy participants (control group). • Inclusion criteria: Symptomatic individuals were selected and these criteria were: recurrent abdominal pain, unexplained vomiting and weight loss (17). • Exclusion criteria: Gastritis patients, gastric CA, history of PUD, patients receiving antibiotics, H2 receptor antagonists or anti-acid treatment, were excluded (17). After informed consent was obtained from all participants, full-mouth examination was performed foreach patient; saliva and gastric sampleswere collected from cases (whom diagnosed endoscopicaly by a specialist) and from healthy subjects (endoscopicaly no signs of PUs disease). Subjects were investigated for the presence of H. pylori in saliva and stomach biopsies, (18). Helicobacter pylori detected in gastric biopsies by histological examination, by using Hematoxylin and Eosin stain (H & E), whilePolymerase Chain Reaction (PCR) was carried out on the saliva.The lab work for the detection of H. pyloriwas done in a strictly asepsis conditions. DNA extraction: DNA was extracted from the saliva using QIAamp® DNA Mini kit (Germany) with a bacterial DNA extraction protocol (Spin Protocol): Samples preserved in freeze thawed into room temperature then 200 μlsaliva pipeted in to a microcentrifuge tube, then20 μl of proteinase K added then200 μllysis buffer (buffer AL) added to the mixture, thenthe mixture incubated at 56°C for 10 min.The mixture was then combined with 200 μl Ethanol and mixed by pulse-vortexing. The mixture was applied to the QIAamp Mini spin column which holds a silica gel membrane, and spun at 8000 r/m (round/minute) for 1 minthen the spin column washed with 500 μl of buffer AW1 and then AW2 by centrifugation at 14,000 r/mfor 1 and 3 minutes respectively, pure DNA bounded on a membrane eluted by 200 μl of buffer AE added and centrifuged for 1 min, then incubated at room temperature (15-25°C) for 5 minute. Finally the resulting DNA extracts stored at -20°C until PCR assessment. DNA amplification and gel electrophoresis: Extracted DNA from saliva samples were amplified by using primerfor urease gene ure C (136 bp) 5' – AAGCTTTTAGGGGTGTTAGGGGTTT – 3' and 5' – CGCAATGCTTCAATTCTAAATCTTG – 3' indicative of H. pylori infection, which have been shown previously to be highly specific and sensitive, (19), and by using GoTaq® Green Master Mix kit (a premixed ready-to-use solution containing bacterially derived Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient amplification of DNA templates by PCR), the GoTaq® Green Master Mix thawed into room temperature then the reaction mixture prepared in a final volume of 50 μl. then the reactions placed in a thermal cycler.DNA amplification was carried out as follow: Denaturation at 94°C for 5 minutes in the first cycle, followed by annealing for 30 seconds at 60°C, extension for 2 minutes at 72°C, and denaturation for 30 seconds at 94°C for a total of 40 PCR cycles.The extension for the last cycle was increased to 5 minutes to ensure complete extension of the amplified fragment. The PCR products were resolved by 2% agarose gel electrophoresis and were visualized after ethidium bromide (0.5 mg/ml) staining, using an UV transilluminator and photographed by Polaroid camera. RESULTS The present study was carried out on 50 patients, 29 males and 21 females, their age ranged from (15-65) years. Among study groups (50) samples from oral cavity (saliva) and (50) samples from stomach (gastric biopsy) from the same individuals were collected, H. pylori was detected in 84% patients simultaneously in both types of samples. This study reported that there was an excellent agreement between detecting H. pylori simultaneously in both stomach and mouth {K > 0.70, P < 0.05, Table 1 A}. If we screen for stomach H. pylori through detecting this microorganism in the mouth; saliva samples is highly sensitive (98%) but not very specific (70%) (Table 1B). J Bagh College Dentistry Vol. 27(3), September 2015 Establishment of the Oral Diagnosis 87 Table 1: Results of detection of H. pylori in gastric and saliva samples: A) Results of statistical tests Statistical Test Output Fisher's exact Test P value < 0.001 Measurement of Agreement Kappa = 0.735 P < 0.001 B) Results of diagnostic values if using oral sample to screen for H. pylori in stomach. Indicator of Test Performance P value 95%CI [Upper; Lower] Sensitivity 0.98 [0.86; 1.00] Specificity 0.71 [0.30; 0.95] Accuracy 0.94 [0.82; 0.98] PPV 0.95 [0.83; 0.99] NPV 0.83 [0.36; 0.99] Also the current study found that presence of H. pylori in saliva was significantly associated withgingival inflammation and halitosisP < 0.05. But no significant associations with teeth erosion, burning mouth syndrome (BMS), oral aphthous P > 0.05. Also there was highly significant correlation between presence of H. pylori in saliva and PUD group P < 0.001 (Figure 1). Figure 1: Distribution of study sample according to study group and to presence of H. pylori in saliva. DISCUSSION In this study, H. pylori was identified simultaneously in both kinds of samples (gastric biopsy and in saliva) for 84% participants.The present findings showed statistical significance of the positive relation of H. pylori in gastric biopsiesand oral samples, these results coincide with that reported by Miyabayashi (20) and Rasmussen (13), indicating that in many cases, patients with positiveresults of gastric biopsies were also positive in oral samples. In this study, the H. pylori saliva test had a high sensitivity 98%, which enabled it to detect a low titer of H. pylori, but it's not very specific 71%, this finding agree with a study reported by Song (21). Also the finding showed particular attention is paid to the association of the H. pylori reservoir in the oral cavity with gingivitis, this finding agrees with Cellini (22). It is also suspected that the presence of this bacteria in PUD patients may lead to oral inflammation and the formation of gingival/periodontal pockets, which develop favorable conditions for bacterial growth (23).The findings of this study support the concept of a potential association between halitosis and the presence of H. pylori in the oral cavity, this findings is highly similar to that reported by Moshkowitz (24) and Suzuki (25). The finding show no significant association between H. pylori presence in saliva and dental erosion, this may be explained by the fact that dental erosion doesn’t occur until gastric acid had acted on the dental hard tissue regularly over a period of several years. In agreement with studies of other researchers (26,27). The present study showed no significant relation between H. pylori and burning mouth syndrome.At variance to other trial, reported by Idan and Abdul-Razaq (28), found highly statistical significant relationship between H. pylori and burning mouth. The findings suggest that salivary H. pylori does not play a role in the pathogenesis of RAU, in agreement with Ghanaei (29) found that these bacteria not involved as a cause of recurrent oral aphthous ulcers because H. Pylori DNA could not be found in the aphthous ulcers of these patients. In conclusion there is strong relation between presence of H. pylori in stomachand oral cavity, itwas identified simultaneously in both kinds of samples, in gastric biopsy histologically and in saliva by PCR.The successful detection of H. pylori DNA directly from saliva samples indicates that this approach is feasible and saliva could serve as an effective and valuable noninvasive specimen to diagnose and monitoring the efficacy of therapy in patients with active H. pylori infection. This method not only reduces the economic burden of treatment, but also lowers the risk of increasing the resistance of H. pylori to antibiotics. P er ce n t % J Bagh College Dentistry Vol. 27(3), September 2015 Establishment of the Oral Diagnosis 88 REFERENCES 1. Majumdar D, Bebb J, Atherton J. Helicobacter pylori infection and peptic ulcers. Medicine 2007; 35: 204-9. 2. Silva MIG, de Sousa FCF. Gastric Ulcer Etiology, Peptic Ulcer Disease. Part 1.China, 2011. 3. Tulassay Z, Herszenyi L. 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