Study on characteristics of caga and vaca genotypes of helicobacter pylori and histopathological lesions of chronic gastritis of ethnic minorities in Viet Nam

Journal of military pharmaco-medicine n o 1-2020 151 STUDY ON CHARACTERISTICS OF cagA AND vacA GENOTYPES OF HELICOBACTER PYLORI AND HISTOPATHOLOGICAL LESIONS OF CHRONIC GASTRITIS OF ETHNIC MINORITIES IN VIETNAM Bui Chi Nam1; Vu Van Khien2; Phan Quoc Hoan2 Duong Xuan Nhuong3; Dao Truong Giang3; Nguyen Thi Loan3 SUMMARY Objectives: To identify characteristics of cagA, vacA genotypes of H. pylori and chronic gastritis in some ethnic minorities in 2 provinces of Dak Lak and Lao Cai, Vietnam. Subjects and methods: 328 patients with clinical manifestations of gastric disease and chronic gastritis on histopathology. Gastrointestinal endoscopy identifies H. pylori infection, biopsy tissue were taken to assess chronic gastritis lesions according to updated Sydney standards; PCR to determine the cagA and vacA genotypic characteristics of H. pylori. Results: The prevalence of Western cagA genotype in Dak Lak accounted for 77.1%, of which Ede people accounted for 82.2%. All Western cagA were Ede people, accounting for 74/90 patients (82.2%) and only 16/90 patients (17.8%) of East Asia cagA. Positive percentage of vacA: 169/169 patients (100%), of which vacA s1m1 and vacA s1m2 accounted for 66.3% and 33.7%, respectively. There was no statistically significant difference in chronic gastritis lesions on histopathology between H. pylori and vacA m1 and m2 strains. Conclusion: Western cagA genotype was dominant in Ede people and the level of chronic atrophy of Western cagA genotype was higher than in East Asia cagA group. * Keywords: Chronic gastritis; Stomach pathology; Helicobacter pylori; Lao Cai; Dak Lak. INTRODUCTION Helicobacter pylori (H. pylori) has now been confirmed as a major cause of chronic gastritis and gastroduodenal ulcer [1]. A number of multinational studies showed that different races have different rates of infection. The blacks have twice as much infection rates as the whites, and this rate also changes when other living conditions and different regions in poor and developing countries have higher rates of infection than developed countries [4]. In Vietnam in recent years, there have been many studies that have clarified many characteristics of H. pylori infection, however, most of studies so far have focused on the Kinh people. 1. Lao Cai General Hospital 2. 108 Military Central Hospital 3. 103 Military Hospital Corresponding author: Dao Truong Giang (giangle127@gmail.com) Date received: 28/11/2019 Date accepted: 11/01/2020 Journal of military pharmaco-medicine n o 1-2020 152 There are only some preliminary studies on the epidemiology of H. pylori infection among ethnic minority children in the Northern border areas [2]. On the other hand, on the study of pathogenicity of bacteria, the authors showed that, among 80% of individuals infected with H. pylori, there were no symptoms and no lesions at all [5], this was because virulence and pathogenicity factors of H. pylori strains may not be the same [6]. Studies on H. pylori in upland ethnic minorities are few and not large-scale, limited and especially research on ethnic minorities has not been mentioned. In order to contribute to the understanding of the above problems, we conducted this study: To analyze the characteristics of cagA, vacA genotype of H. pylori bacteria and characteristics of chronic gastritis of ethnic minorities humans in Vietnam. SUBJECTS AND METHODS 1. Subjects. The subjects were determined to identify chronic gastritis (CG) based on histopathological results in two provinces: Lao Cai with 198 patients, Dak Lak with 130 patients, from March 2013 to June 2014. * Selection criteria: - Patients ≥ 18 years old, regardless of gender or occupation. - Diagnosed with CG based on histopathological results. - Never had stomach surgery, no stomach cancer or stomach-duodenal bleeding. - Did not use drugs to treat H. pylori eradication or use antibiotics, antacids, bismuth components within 1 month before participating in the study. 2. Methods. * Study design: cross-sectional description. * Research methods: Means and materials for research: Gastro-duodenum scope machine GIF-160, GIF-180 (Olympus, Japan); biopsy forceps; vial of solution contains 10% neutral formol to immobilize tissue samples, and media for culture, PCR test, H. pylori gene sequencing. * Steps to proceed: - Having interviewed with the form of study plan. - Having conducted endoscopy and biopsy: Gastroscopy according to the endoscopic procedure issued by the Ministry of Health, conducting an 8-piece biopsy to do clo-test, histopathology and PCR. - All collected samples will be stored properly, packed, transported and tested at Oita University (Japan). * Histopathological examination: Diagnosis of CG by histopathology according to the updated classification criteria of the Sydney classification system [7], conducted by Dr. Tomohisa Uchida (Oita University, Japan). Results were evaluated independently based on patient code. Diagnosis of H. pylori by clo-test, histopathology, culture of H. pylori and PCR. - H. pylori containing East Asia cagA type includes H. pylori strains containing EPIYA ABD or ABDD. - H. pylori containing Western cagA type includes H. pylori strains containing EPIYA ABC, ABCC or ABCCC. - H. pylori contains indeterminate cagA when cagA strain only contains EPIYA AB. Journal of military pharmaco-medicine n o 1-2020 153 RESULTS AND DISCUSSION We collected 328 patients with 169 H. pylori (+) samples from culture (Lao Cai: 73 samples, Dak Lak: 96 samples) to conduct an analysis of cagA (+) ratio and subtype of H. pylori's cagA. Table 1: Ratio and distribution of cagA genotypes of H. pylori. Category cagA (+) H. pylori (+) cagA (+) n (%) cagA Western, n (%) cagA East Asia, n (%) Lao Cai (n = 73) 73/73 (100%) 73/73 (100%) Mong 37/37 (100%) 37/37 (100%) Nung* 2/2 (100%) 2/2 (100%) Tay* 12/12 (100%) 12/12 (100%) Dao * 9/9 (100%) 9/9 (100%) Xa Pho 11/11 (100%) 11/11 (100%) Ray 1/1 (100%) 1/1 (100%) Man 1/1 (100%) 1/1 (100%) Dak Lak (n = 96) 96/96 (100%) 74/96 (77.1%) 22/96 (22.9%) E De 90/90 (100%) 74/90 (82.2%) 16/90 (17.8%) Nung 4/4 (100%) 4/4 (100%) Van Kieu 1/1 (100%) 1/1 (100%) Thai 1/1 (100%) 1/1 (100%) Total (n = 169) 169/169 (100%) 74/169 (43.8%) 95/169 (56.2%) Positive cagA: 169/169 patients (100%), of which Western cagA accounted for 74/169 patients (43.8%), East Asia cagA accounted for 95/169 patients (56.2%). In Lao Cai: The rate of cagA was positive: 73/73 patients (100%) and all ethnic groups in Lao Cai had East Asia cagA (100%). In Dak Lak: Positive cagA: 96/96 patients (100%), of which Western cagA: 74/96 patients (77.1%), East Asia cagA: 22/96 patients (22.9%). Especially, all Western cagA were Ede people, accounting for 74/90 patients (82.2%) and only 16/90 patients (17.8%) of East Asia cagA. Other ethnic groups had East Asia cagA. Other ethnic groups (Nung, Thai, Van Kieu: total of 6 patients) in Dak Lak all had East Asia cagA. The Western cagA in Dak Lak accounted for 76.8%, of which Ede people accounted for 82.2%. Ho Dang Quy Dung [3] collaborated with Oita University (Japan) to determine the prevalence of H. pylori infection and identify the virulence factors of H. pylori in CG patients (n = 242) in Kinh people in Hochiminh City and Hanoi. The results showed that the rate of East Asia cagA, Western cagA and the negative cagA accounted for 91.3%, 1.9% and 3.7%, respectively. The results also showed that Journal of military pharmaco-medicine n o 1-2020 154 the rate of East Asia cagA in Hanoi was 94.4%, in Hochiminh City was 87.8%, the difference was not significant (p > 0.05). The proportion of H. pylori strains with positive cagA was very high in East Asian countries such as Korea, Japan (approximately 100%) [8], while in other regions it was much lower [6]. A study in Italy by Paoluzi O.A et al found that the ratio of H. pylori carrying cagA was 60.7% [9]. Our study showed a positive cagA rate of 100%. However, East Asia cagA accounted for only 56.2%, while Western cagA accounted for 43.8%. Especially, in Ede people the Western cagA rate accounted for 82.2%. Studies showed that in countries with high rates of cagA, especially in East Asian cagA, those countries had high rates of gastric cancer (GC) [6]. Table 2: The ratio of vacA, vacA s1m1 and vacA s1m2 of H. pylori. vacA s and m Culture H. pylori (+) vacA positive s1m1 s1m2 Lao Cai (n = 73) 73/73 (100%) 34/73 (46.6%) 39/73 (53.4%) Mong 37/37 (100%) 16/37 (43.2%) 19/37 (51.4%) Nung* 2/2 (100%) 2/2 (100%) Tay* 12/12 (100%) 5/12 (41.7%) 7/12 (58.3%) Dao * 9/9 (100%) 4/9 (44.4%) 5/9 (55.6%) Xa Pho 11/11 (100%) 4/11 (36.4%) 7/11 (63.6%) Ray 1/1 (100%) 1 (100%) Man 1/1 (100% ) 1 (100%) Dak Lak (n = 96) 96/96 (100%) 78/96 (81.2%) 18/96 (18.8%) Ede 90/90 (100%) 74/90 (82.2%) 16/90 (17.8%) Nung* 4/4 (100%) 2/4 (50.0%) 2/4 (50.0%) Van Kieu 1/1 (100%) 1 (100%) Thai 1/1 (100%) 1 (100%) Total (n = 169) 169/169 (100%) 112/169 (66.3%) 57/169 (33.7%) The ratio of positive vacA: 169/169 (100%), of which vacA s1m1 and vacA s1m2 accounted for 66.3% and 33.7%, respectively. In Lao Cai: The rates of vacA s1m1 and vacA s1m2 accounted for 46.6% and 53.4%, respectively. In Dak Lak: vacA s1m1 and vacA s1m2 accounted for 81.2% and 18.8%, respectively. There is a close relationship between bacterial toxins and H. pylori pathogen, of which the type of vacA s1m1 was considered to be the most virulent. The prevalence of vacA s1m1 appeared mainly in Ede ethnic minority patients in Dak Lak and this was a risk factor leading to the formation of various gastric Journal of military pharmaco-medicine n o 1-2020 155 pathologies. However, the opposite was that the proportion of Western cagA was high (82%), while that of East Asia cagA accounted for only 18%. Ho Dang Quy Dung showed that vacA s1 accounted for 100% and vacA s1m1, vacA s1m2 accounted for 44.6% and 51.5%, respectively; there were 4 strains with unidentifiable genotype, accounting for 3.9%. The results also showed that the percentage of vacA m1 in Hanoi patients group (57.7%) was higher than vacA m1 in Hochiminh City (34%), the difference was statistically significant (p < 0.05) [3]. Studies abroad conducted on different races, in different geographical regions, also showed differences in the rates of vacAm1 and vacAm2. The research by Basso D et al showed vacA s1 ratio was 57.6%, vacA m1 was 40%, vacA s1 ratio was significantly lower than our results (p < 0.05). Another similar study by Caner V et al showed that vacA s1 accounted for 89.1%, vacA m1 17.4% [10]. In the world, studies of cagA and vacA ratios in CG patients had different and conflicting data - conflicting explanations about the formation of different stomach diseases. India has a very low rate of GC (4.7 for men and 2.9 for women/100,000 people). However, in the study by Kumar S et al, 86.9% of H. pylori strains had the cagA gene, 100% of strains had vacA s1 and 80% of strains had vacA m1, a very high percentage. As such, these parameters are very contrary to the incidence of GC and this is very difficult to explain. A study by Lui S.K et al by sequencing the cagA gene of H. pylori strains from Indians showed that the ratio of EPIYA-ABC motif was 46.7%, EPIYA-ABCC was 33.3%, 6.7% was EPIYA-AC (a variant of Western type cagA) and only 13.3% had EPIYA- ABD cagA type East Asia) [11]. Rhead et al identified an important region in the vacA gene, which is located between the region s and the region m and is named the region i (intermediate region). Region i is divided into i1 (with vacuoles) and i2 (there is no vacuoles). For the Western research group, all vacA s1/m1 have i1, all vacA s2/m2 have i2, and vacA s1/m2 can be i1 or i2 [12]. Table 3: Relationship between atrophic inflammation, intestinal dysplasia and cagA type. Histopathology East Asia cagA (n,%) Western cagA (n, %) Total (n, %) Mild atrophy 41/89 (46.1%) 48 (53.9%) 89 (84.0%) Moderate atrophy 9 (60.0%) 6 (40.0%) 15 (14.2%) Severe atrophy 2 (100%) 0 (0%) 2 (1.9%) Intestinal dysplasia 16 (69.6%) 7 (30.4%) 23 (100%) p < 0.05 There was a correlation between atrophy, intestinal dysplasia and cagA type with p < 0.05, of which, the percentage of mild atrophy and cagA East Asia accounted for 46.1%, Western cagA type accounted for 53.9%. The rate of moderate atrophy and cagA East Asia type accounted for 60.0%, Western cagA type accounted for 40.0%. Journal of military pharmaco-medicine n o 1-2020 156 The rate of severe atrophy and type cagA East Asia accounted for 100%, Western cagA type accounted for 0%. The rate of intestinal dysplasia and cagA East Asia accounted for 69.6%, the Western cagA type accounted for 30.4%. Table 4: Relationship between vacA m types and histopathological lesions. vacA m gene type Level assessment scale/histopathology Grade m1 m2 p Non 30 (26.8%) 22 (37.3%) Mild 55 (49.1%) 26 (44.0%) Moderate 24 (21.4%) 5 (4.5%) H. pylori density Severe 3 (2.7%) 6 (10.2%) < 0.05 Non 38 (33.9%) 27 (45.7%) Mild 66 (58.9%) 23 (39.0%) Moderate 8 (7.2%) 7 (11.9%) The degree of atrophy Severe 0 2 (3.4%) < 0.05 Non 88 (78.6%) 41 (69.5%) Mild 20 (17.9%) 16 (27.1%) Moderate 1 (0.9%) 1 (1.7%) Dysplasia level Severe 3 (2.7%) 1 (1.7%) > 0.05 There was no statistically significant difference in CG lesions on histopathology between H. pylori and vacA m1 and m2 strains. Table 5: Relationship between cagA type and location of gastric lesions through histopathology. Type cagA Level assessment scale/histopathology Location East Asia Western p Antral 79 (79.8%) 58 (78.4%) > 0.05 The degree of neutrophil leukocyte penetration Body 39 (39.4%) 43 (58.1%) < 0.05 Antral 62 (62.6%) 72 (97.3%) < 0.05 The degree of leukocyte penetration Body 28 (28.3%) 31 (41.9%) > 0.05 Antral 48 (48.5%) 53 (71.6%) < 0.05 Atrophic inflammation Body 4 (4.0%) 1 (1.4%) > 0.05 Total 99 (100%) 74 (100%) Journal of military pharmaco-medicine n o 1-2020 157 Penetration of neutrophils, mononucleosis and atrophic inflammation were significantly higher in Western cagA patients than in East Asia cagA group in both antrum and body (p < 0.05). Particularly, the penetration level of neutral balance board in antral was not different between the two groups. Histopathological changes in patients with CG were related to the causative agent, of which H. pylori played an important role. Studies in Japan, Korea and China clearly demonstrated the role of cagA gene, especially with East Asia cagA gene. According to Yamaoka Y et al, a study in Okinawa (Japan) showed that the level of active chronic inflammatory disease (IUD) and atrophic inflammation significantly increased in H. pylori infections with East Asian cagA type compared to H. pylori infection patients was negative cagA or Western type cagA. Statistics on the island of Okinawa (Japan) showed that the incidence of GC in this place was significantly lower than the frequency of GC in the country. It can be explained, since the World War II, there were many Americans living on this island. Interferences of human race and environmental conditions, the occurrence of Western cagA gene more than Eastern cagA... caused changes in the frequency of low rate of GC in this region [13]. Similarly, studies in Thailand indicated that the incidence of GC was closely related to the prevalence of Eastern Asia and Western cagA. The prevalence of GC in Thailand was very low (2.9/100,000 people) and was classified as low prevalence of GC. The results showed that the rate of East Asia cagA accounted for less than 50% and the rest was mostly Western type [14]. CONCLUSION - There was a big difference in the ratio of Western cagA in Dak Lak and Lao Cai, the Western cagA in Dak Lak accounted for 77.1%, of which Ede people accounted for 82.2%. All Western cagAs were Ede people, accounting for 74/90 patients (82.2%) and only 16/90 patients (17.8%) of East Asia cagA. Positive percentage of vacA: 169/169 patients (100%), of which vacA s1m1 and vacA s1m2 accounted for 66.3% and 33.7%, respectively. In Lao Cai: The rates of vacA s1m1 and vacA s1m2 accounted for 46.6% and 53.4%, respectively. In Dak Lak: rates of vacA s1m1 and vacA s1m2 accounted for 81.2% and 18.8%, respectively. - There was a relationship between the degree of atrophy, intestinal dysplasia and type cagA. Penetration rates of neutrophils, mononucleosis and atrophic inflammation were significantly higher in Western cagA patients than in East Asia cagA group in both antrum and body. * Recommendation: There should have other studies to analyze the polymorphism of cagA, vacA virulence factors and find out about host factors in CG patients in order to clearly assess the role of different virulence factors, the clinical consequences need to be evaluated based on the combination of virulence factors rather than the evaluation of each individual factor. Journal of military pharmaco-medicine n o 1-2020 158 REFERENCES 1. Đào Văn Long. Bài tiết axít dịch vị và bệnh lý liên quan. Nhà xuất bản Y học. Hà Nội. 2014, tr.60-154. 2. Mai Hồng Bàng. Đặc điểm lâm sàng, hình ảnh nội soi và mô bệnh học của ung thư dạ dày. Tạp chí Y học Thực hành. 2006, 3, tr.87-89. 3. Hồ Đăng Quý Dũng và CS. Nghiên cứu mối liên quan giữa các yếu tố độc lực cagA, vacA của Helicobacter pylori với viêm dạ dày mạn tính. Tạp chí Y Dược Lâm sàng 108. 2011, 6, tr.60-67. 4. Ferlay J, S.I, Dikshit R et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. J Cancer. 2015. 136 (5), pp.E359-E386. 5. Japanese Gastric Cancer Association. Japanese classification of gastric carcinoma: 3rd English edition. Gastric Cancer. 2011, 14, pp.101-112. 6. Azuma T. Helicobacter pylori cagA protein variation associated with gastric cancer in Asia. Journal Gastroenterol. 2004, 39 (2), pp.97-103. 7. Dixon M. F, Path F.R.C, Genta R.M et al. Classification and grading of gastritis: The updated Sydney system. American Journal of Surgical Pathology. 1996, 20 (10), pp.1161-1181. 8. Asaka M et al. Atrophic gastritis and intestinal metaplasia in Japan: Results of a large multicenter study. Helicobacter. 2001, 6, pp.294-299. 9. Paoluzi O.A et al. Discrepancy between polymerase chain reaction assay and Western blot analysis in the assessment of cagA status in dyspeptic patients. Helicobacter. 2001, 6, pp.130-135. 10. Caner V et al. H. pylori iceA alleles are disease-specific virulence factors. World J Gastroenterol. 2007, 14, pp.2581-2585. 11. Lui S.Y, Chuah S.W et al. Different cagA and vacA polymorphisms are found in the Chinese versus the Malay and Indian populations: An analysis of Helicobacter pylori virulence genes in Singapore. Proceedings of Singapore Heathcare. 2010, 19, pp.12-18. 12. Choi S, Lim Y.J, Park S.K. Risk factor for metaplastic gastritis of Koreans. World Journal of Gastroenterology. 2006, 12 (16), pp.2584-2587. 13. Yamaoka Y, Kato M, Asaka M. Geographic differences in gastric cancer incidence can be explained by differences between Helicobacter pylori strains. Internal Medicine. 2008, 47 (12), pp.1077-1083. 14. Vilaichone R.K, Mahachai V et al. Molecular epidermiology and outcome of Helicobacter pylori infection in Thailand: A cultural cross roads. Helicobacter. 2004, 9, pp.453-459.