Detection of oral Helicobacter Pylori infection using saliva test cassette

Objective: To investigate the incidence of oral infection with Helicobacter pylori (H. pylori) and identify related epidemiological factors among freshmen of four colleges in Yancheng. Methods: The data, scored positive or negative, were collected on 160 individuals who had been diagnosed by H. pylori Saliva Test Cassette (HPS) during October 2013 to October 2014. H. pylori Saliva Test Cassette (HPS) is to use colloidal gold technique to specifically identify urease in saliva. A standard questionnaire, with variables including sex, educational degree of parents etc., was used in the subjects. Statistical data of diagnostic test were analyzed by SPSS17.0 software. Results: Out of 160, 82 subjects were detected positive and 78 were negative. In univariate analysis, dental plaque, family history of stomach diseases, habit of washing hands before meals and habit of brushing teeth twice daily were associated negatively with H. pylori infection. Multivariate logistic regression analysis showed that dental plaque and family history of stomach diseases were the risk factors which may be associated with H. pylori infection. Conclusions: Dental plaque and family history of gastric diseases were risk factors of oral H. pylori infection. It is vital for the prevention of H. pylori infection to focus on health education and oral hygiene, and avoid transmission by oral-oral route as well.


INTRODUCTION
It has attracted extensive attention that Helicobacter pylori infection correlates closely to chronic gastritis, peptic ulcer and gastric cancer. 1,2 H. pylori were considered as group 1 carcinogen by the International Agency for Research on Cancer (IARC) in 1994. Recently, researchers have discovered that there may be some relativity between H. pylori infection and many extragastro intestinal diseases, such as hematological, skin, and cardiovascular and respiratory system diseases. [3][4][5][6] Diagnostic tests of H. pylori infection include invasive and noninvasive methods. Noninvasive tests such as immunoassay for serological antibodies against H. pylori, 13 C Urea Breath Test and 14 C-Urea Breath Test (UBT), are more acceptable. Traditional serological test mainly detects the specific IgG antibody against H. pylori in serum, but can't distinguish current infection from previous infection. 7 UBT mainly identifies H. pylori infection in gastric mucosa by determining the amount of 13 CO 2 or 14 CO 2 in breath, by means of measuring equipment with high sensitivity after subjects intake a certain amount of 13 C or 14 C-labeled urea. [7][8] Saliva H. pylori antibody testing and stool antigen testing can readily be manipulated, as many studies have reported. 9,10 However, it was seldom reported regarding saliva H. pylori antigen testing. 11 H. pylori Saliva Test Cassette (HPS) is to use monoclonal antibody to specifically identify urease in saliva after H. pylori infection. Its sensitivity is 10ng/mL. Although many bacteria in oral cavity can produce urease, common oral bacteria were analyzed and did not show interference or cross-reactivity with the test. A number of researches have compared HPS to UBT/RUT, 12,13 and shown that there is a correlation between results of 13 C-UBT and HPS. Combination of 13 C-UBT and HPS may compensate for the blind zone of 13 C-UBT detecting oral H. pylori infection. 13 C-UBT should not be a gold standard to judge the validation of HPS in detecting oral H. pylori infection. HPS is a new noninvasive method, which is especially applicable to detect oral H. pylori infection.
In order to investigate the state of oral infection with H. pylori and identify the correlation between H. pylori infection and epidemiological factors (e.g., eating habits, oral hygiene and disease history, etc.) among students of four colleges in Yancheng, we utilized H. pylori Saliva Test Cassette to test 160 newly admitted patients and administrated a questionnaire. We expect to improve the knowledge of H. pylori infection among youngsters, help those with high risk factors attach importance to periodic checks, and provide basis for prevention programs as well.

METHODS
Subjects were provided with a written information letter about the study who gave their informed consent.Ethics committee of Yancheng Institute of Health Sciences approved our study design, methods and the consent procedure used for this study which was conducted during October 2013 to October 2014. Subjects: Using stratified random sampling method, we randomly selected 160 fresh students (69 male and 91 female, mean age 18.38±0.85) enrolled in 2013 from the four colleges in Yancheng as respondents. They were free from medication with PPIs or histaminereceptor antagonists for a minimum of 2 weeks and antibiotics or colloidal bismuth subcitrates for 4 weeks prior to testing. 7 Detection method: The H. pylori Saliva Test Cassette (Ameritek Diagnostic Reagent Co., Ltd, Jiaxing, China) was used to detect the oral infection of H. pylori. The subjects were not allowed to brush their teeth, rinse their mouths, drink water or take foods one hour prior to the test. A minimum of 0.5 mL of saliva was collected into a disposal testing cup each and the test was performed within 5 minutes. According to the manual, we added the mixture of saliva and buffer solution into the sample well, and observed the result within 20-30 min. As the test kit began to work, red color was seen to move across the result window in the center of the test disk. The presence of 2 red color bands ('T' band and 'C' band, i.e., testing band and control band) within the result window indicated a positive result. The presence of only 1 color band indicated a negative result. If there was no control band, the sample should be retested. 13 Investigation method: A standard questionnaire was administrated to the subjects. Characteristics include sex, educational degree of parents (university or less than university degree), frequency of eating out weekly (≥4 or <3), consumption of fresh vegetables and fruits (daily or not daily), habit of hand washing before meals (frequent or seldom), brushing habit (twice daily or once daily), dental plaque, dental caries and family history of stomach diseases. The diagnoses of dental plaque and dental caries were established on the basis of entrance physical examination. Family history of stomach diseases included chronic gastritis, peptic ulcer and gastric cancer diagnosed by clinical practice doctors. All the 160 subjects signed informed consents and completed the investigation. Statistical analysis: Data analysis was conducted using SPSS 17.0 software. A univariate analysis was conducted to explore how each characteristic is associated with H. pylori infection. Statistical significance was determined using Chi-square tests and was defined as a p-value of <0.05. We included variables that were significant in univariate analysis to conduct a multivariate logistic regression analysis. The regression model was using "Enter" method. The inclusion criteria were 0.05 and the exclusion criteria were 0.10.

RESULTS
A total of 82 subjects were detected positive and 78 were negative. The oral H. pylori infection rate among them was 51.25%. Original data were scored positive and negative. Other data are all listed in  students. At first, serological tests and stool tests were taken into account. But we excluded serological tests due to its less accuracy. 7 As regards stool antigen tests, we were worried about the decline of sensitivity with samples left at room temperature. 14 The method of UBT has always been considered as the gold standard to judge H. pylori infection, to judge H. pylori infection in stomach, based on its principle. The method of HPS has the advantage of being economical and simplicity, moreover, its specificity and sensitivity in detecting H. pylori in oral cavity had been confirmed. 13 Positive results of HPS can indicate the occurrence of oral infection alone or co-infection of gastric and oral H. pylori.
In previous publications regarding the detection of H. pylori infection in dental plaque and saliva, clinical patients were recruited as study population 15,16 and there were no references pertaining to the oral infection status in the general population. The oral H. pylori infection rate among our participants was 51.25%.
Our study didn't find that regular consumption of fresh fruits and vegetables can reduce the risk of oral H. pylori infection, although several studies have suggested high intake of fresh fruits and vegetables may reduce the risk of gastric cancer. 17,18 As Herrera thought, a risk of transmitting H. pylori to humans may be caused by intake of contaminated vegetables. 19 Our study also showed that good hygiene habits, like washing hands before meals and brushing teeth twice daily, played a role in the prevention of oral H. pylori infection.
In the present study, dental plaque and family history of gastric diseases were risk factors of oral H. pylori infection, which suggested that oral diseases may have correlation with H. pylori infection and there existed a familial clustering phenomenon of H. pylori infection. Our findings extend the evidence from previous reports that oral-oral transmission may be a primary route of H. pylori infection. 20 To minimize the risk of H. pylori infection, it is recommended to change poor health habits and pay attention to dietary hygiene and oral hygiene. What's more important is to see the dentist to remove dental plaque and calculus in time, and to separately eat by dishes to avoid cross infection by fecal-oral or oral-oral transmission among family members. 21 Our students may also strengthen health education in their families and communities. Since the possibility of infection developing into peptic ulcer and gastric cancer will continue to be a critical problem in the future, 22 those who have gastrointestinal symptoms with positive HPS results require further testing of gastric H. pylori infection. If there are signs of H. pylori infection in stomach, treatment is needed. Moreover, since H. pylori colonized in oral cavity might be one of the major causes of the recurrence after eradication therapy, 23 it is worthwhile to note that we should eradicate H. pylori in oral cavity at the same time when preventing recurrence.

CONCLUSIONS
H. pylori Saliva Test Cassette was used to detect the oral H. pylori infection rate and results showed that dental plaque and family history of gastric diseases were risk factors of oral H. pylori infection. Taking the possibility of inducing gastric infection into account, we need to establish and keep good hygiene habits, especially oral hygiene, and avoid cross infection among family members.