The organization of Helicobacter pylori cag-pathogenicity island (cagPAI) genes in multiracial population with histopathological changes of gastric mucosa

The organization of Helicobacter pylori cagpathogenicity island (cagPAI) genes in multiracial population with histopathological changes of gastric mucosa Al zah Hana ah (  al zah@ppukm.ukm.edu.my ) Universiti Kebangsaan Malaysia Fakulti Perubatan https://orcid.org/0000-0003-4859-1439 Shaza Azlin Razak University Kebangsaan Malaysia Hui-min Neoh Universiti Kebangsaan Malaysia Noraziah Mohamad Zin Universiti Kebangsaan Malaysia Bruno S. Lopes University of Aberdeen Research article Keywords: Helicobacter pylori, cagPAI, histopathological scores, multi-ethnic population Posted Date: May 14th, 2019 DOI: https://doi.org/10.21203/rs.2.9601/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/15 Abstract Background: Helicobacter pylori is a Gram-negative bacillus that colonises only the mucus layer of the human stomach and is implicated in gastric diseases. Virulent H. pylori harbouring cag-pathogenicity island (cagPAI) which encodes genes for type IV secretion system (T4SS) and CagA protein is one of the major virulence determinants involved in disease development. We examined the entire cagPAI genes in 95 H. pylori isolates from a multiracial population and examined the intactness of cagPAI region with histopathological scores of the gastric mucosa. Results: 95.8% of H. pylori isolates were cagPAI-positive with 23.2% having an intact cagPAI, whereas 72.6% had a partial/rearranged cagPAI. In our study, cag2 and cag4 were found to be signi cantly higher in H. pylori isolated from Malays, whereas cag4 was predominant in Chinese isolates. We also detected cag24 in signi cantly high proportion in isolates from the Malays and the Indians compared to the Chinese isolates. The intactness of cagPAI region showed an association with histopathological scores of the gastric mucosa. Signi cant association was observed between H. pylori harbouring partial cagPAI and higher density of H. pylori and neutrophil activity, whereas strains which lacked cagPAI was associated with higher in ammatory score. Conclusions: The screening of the entire cagPAI genes provides an accurate overview of the cagPAI organisation in H. pylori isolates in a multiracial population. The genotypes of H. pylori strains with various cagPAI rearrangement associated with patients’ ethnicities and histopathological scores might contribute to the pathogenesis of H. pylori infection in a multi-ethnic population. Background Helicobacter pylori is a Gram-negative, microaerophilic, curved-shaped and agellated bacterium frequently found in the stomach of humans [1]. It is an important pathogen that causes gastrointestinal diseases such as chronic gastritis, peptic ulcer, gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma [2,3], although most infected patients appeared asymptomatic. Hence, H. pylori is also classed as type I carcinogen [4]. Factors that contribute to the infected patient’s disease sequelae include environmental factors such as lifestyle and diet , host genetics, host immune responses and bacterial virulence factors [4-6]. Cytotoxin-associated gene pathogenicity island (cagPAI) is one of the major virulence factors associated with disease outcome in infected hosts. It is approximately 40 kb in size consisting of around 28 genes [7], encoding mainly CagA protein, type IV secretion system (T4SS) and other genes for induction of host’s interleukin-8 (IL-8) [7,8]. Although the mechanisms resulting in severe disease development are poorly understood, a major factor is likely to be H. pylori-induced gastric injury and in ammation [9]. Studies show that intactness of cagPAI has a signi cant correlation with disease severity, whereas H. pylori strains with partial deletions within cagPAI region are signi cantly less-pathogenic in nature [10,11]. However, the rates of severe disease development vary between human populations, and differences in H. pylori genotypes may partially explained these differences [12,13]. Page 2/15 Integrity of cagPAI seems to have an important role in the progress of the gastroduodenal disorders, so that intact cagPAI could be seen in H. pylori strains from countries with higher rate of gastric cancer [14]. This integrity also has important effect on the induction of in ammatory response in the gastric mucosa [15]. Several studies have investigated the association of H. pylori cagPAI and gastroduodenal diseases [14,16], however, knowledge about the relationship between H. pylori cagPAI intactness and changes of the infected gastric tissue is sparse. More than 90% of H. pylori strains in Malaysia are cagPAI-positive [17] and Malaysian population consists of multi-ethnic people, therefore the interaction of H. pylori strains with different genotype in various host genetics may have an impact on the differences in disease development. The organisation of cagPAI genes in H. pylori in Malaysian population which has multi-ethnic groups of people has not been well studied. There is lack of comprehensive information with regards to abundance of intact versus rearranged cagPAI among H. pylori strains in this population. Hence, in this study, we sought to characterise the genes within cagPAI and to determine the association of various cagPAI structure in H. pylori isolates with histopathological changes of the infected gastric mucosa. The outcome of this study may provide valuable information in order to draw association between existence of cagPAI genes and its association with disease sequelae in strains from multi-ethnic population and also in strains isolated in different histopathological conditions. Results Histopathological characteristics of the gastric mucosa in the studied populations Histopathological scores of the gastric mucosa among different ethnic groups showed that the Malays had higher mean scores for H. pylori density and neutrophil activity whereas the Chinese showed higher grade of in ammation (Table S1). Higher mean score for intestinal, metaplasia was observed among the Indians, while the atrophy of higher grade was observed in the Chinese. Patients of different ethnicities were grouped into different types of disease conditions based on the histopathological changes (Table S2), i.e chronic gastritis (CG) (n=20), chronic active gastritis (CAG) (n=44) and intestinal metaplasia/atrophy (IM/Atr) (n=28). There was a signi cant difference in the proportion of CG and CAG between the Chinese and the non-Chinese patients. CG was diagnosed more in the Chinese patients compared to the non-Chinese (p = 0.03), whereas CAG and IM/Atr were observed more in the non-Chinese than the Chinese (p = 0.042). Distribution of the cagPAI genes in H. pylori isolates Detection of the cagPAI region in our clinical H. pylori isolates showed that 95.8% (n=91) of the isolates were cagPAI-positive. Four genes in the cagPAI region (cag1, cag6, cag8 and cag21) were detected in all isolates whereas 35.2% isolates were cag2 (n=32) and 52.7% cag14 (n=48) (Table 1). Detection of other Page 3/15 genes ranged from 69.2 – 98.9%. The absence of cag2 was con rmed with 690 or 1100 bp amplicon using empty-site PCR as described by Schmidt et al., [21]. cag14 was detected using 4 sets of primer pair as described by Ta et al., [20]. Six genes (cag1, cag5, cag6, cag8, cag12 and cag26) in the cagPAI region were detected in all Indian isolates, whereas 12 and 19 genes were detected in all Chinese and Malay isolates, respectively (Table 1). A signi cant difference in detection of cag2, cag4, cag14 and cag24 were observed among H. pylori from patients with different ethnicities. Detection of cag2 was signi cantly high in isolates from Malays (86.7%), followed by Indians (57.9%) and was least in Chinese isolates (9.8%) (2 = 36.620, df =2, p < 0.0001). The presence of cag4 was high in isolates from Chinese (80.4%) compared to the Malays (46.7%) and Indians (63.2%) (2 = 7.001, df =2, p = 0.03). Signi cant difference was observed in the detection of cag14 in the Malay isolates (93.3%) compared to the Chinese (39.2%) and the Indian (52.6%) isolates (2 = 13.603, df = 2, p = 0.001). Also, the cag24 was signi cantly higher in the isolates from the Malays (93.3%) and the Indians (89.5%) compared to isolates from the Chinese patients (54.9%) (2 = 12.701, df = 2, p = 0.002). We did further analyses to look for the distribution of individuals cagPAI genes in different disease conditions. All the cagPAI genes show similar distribution in CG, CAG and IM/Atr except for the cag2 (data not shown). cag2 was detected in 15.8% (3/19) of CG, 38.1% (16/42) of CAG and 40.7% (11/27) of IM/Atr. However, no signi cant difference was observed for the detection of H. pylori carrying cag2 in different group of diseases (p = 0.16). Analysis of cagPAI intactness in H. pylori isolates The cagPAI was de ned as intact if all the gene sets of the cagPAI were present including strains lacking only the cag2 (HP0521). A previous systematic mutagenesis study showed that the HP0521 gene was not involved in the process of CagA translocation and IL-8 induction Fischer et al., [7]. In addition, NCBI database de ned the HP0521 as a pseudogene (NCBI-Gene ID: 900040) (DBGET/LinkBD: an integrated database retrieval system, last accessed Oct 8, 2018). Partial cagPAI was de ned when an isolate lacked one (other than HP0521) or more of the cagPAI genes, while negative/deleted cagPAI was de ned if none of the genes were present and a product of approximately 650 bp with primers from the anking regions was obtained. Among the 91 cagPAI-positive H. pylori strains, 24.2% (n=22) had intact cagPAI and 75.8% (n=69) exhibited partial (rearranged) cagPAI. Strains harbouring intact or partial cagPAI were not associated with patients’ ethnicities (p > 0.05). Association between cagPAI intactness and histopathological scores of the gastric mucosa are shown in Table 2. The presence of partial cagPAI was signi cantly related to the higher total score of H. pylori density (p = 0.036) and neutrophil activity (p = 0.03) compared to the intact cagPAI. H. pylori harbouring deleted cagPAI was signi cantly correlated with higher in ammatory score (mononuclear in ltration) compared to H. pylori with partial cagPAI (p = 0.002). The distribution of H. pylori with intact cagPAI was Page 4/15 detected more in the gastric mucosa with IM/Atr, whereas partial cagPAI H. pylori was detected more in CAG, however the difference was not signi cant (Table 3). Discussion Racial differences in the prevalence of H. pylori infection and disease-related severity were observed among patients from multiracial ethnicities [22,23]. Bacterial virulence factor is one of the contributing factors to the development of severe H. pylori-related diseases. The diversity of cagPAI region in the H. pylori genome may have a modifying effect on the pathogenic potential of the infecting strain [24]. In this study, we comprehensively determined the presence of all cagPAI genes in 91/95 H. pylori isolates from Malaysian population which were isolated from patients of different ethnic groups. The results show that more than 95% of our H. pylori strains were cagPAI-positive where 24.2% of the isolates carry all cagPAI genes, 75.8% exhibited partial or rearrangement in the cagPAI genes. In our previous study, we detected only 3.2% of the isolates carrying all the selected cagPAI genes [17]. The low percentage of H. pylori isolates harbouring intact cagPAI genes in our previous study is because we analysed only a subset of the cagPAI genes (cag67, cag10, cag13, cagT, cagM and cagE) as these genes was shown to have linkage between certain genes in the cagPAI region and severe disease as described by earlier studies [25,26]. In contrast, high frequency of intact cagPAI and low frequency of partial cagPAI in H. pylori strains isolated from similar ethnic populations was reported by Schmidt et al., [21]. In their study, few cagPAI genes (cagE, cagL, cagT and HP521) were examined to detect the intactness of cagPAI region. Discordant in the frequency of cagPAI intactness in many reports was due to the difference cagPAI genes that being examined [14,27,28]. Thus, results of the present study indicate that deletions can occur in all parts of the cagPAI and screening the entire genes in the cagPAI is needed to determine the accurate organization of the cagPAI region. For comparison with our results, we reviewed only studies that screened all the cagPAI genes. A previous study observed complete cagPAI present in 82.6% of the strains, while a partially deleted cagPAI in 9.6% of the strains and 7.7% lacked the entire cagPAI in Indian population [11]. In Swedish population, 76% of the strains carried an intact cagPAI, 15% had partially deleted cagPAI and the cagPAI was lacked in 9% of the strains [10]. A study by Azuma et al., [29] showed that the complete cagPAI was identi ed in all 11 Japanese isolates. Variation in the cagPAI positivity in different population of H. pylori isolates might be related to the difference in geographical origin of H. pylori subpopulations. Carriage of the cagPAI region is almost universal presence in H. pylori hpEastAsia and hpAfrica1 populations, intermediate presence in hpEurope and complete absence in hpAfrica2 [19]. Malaysian isolates showed a mixed subpopulation of hpEastAsia, hpAsia2 and hpEurope as indicated by multiracial communities living in the country [30,31]. Analysis of the entire cagPAI genes in the present study revealed that cag1, cag6, cag8 and cag21 were present in all isolates. These genes might represent core genes of the cagPAI region, however function of the cag1, cag6 and cag21 are still unknown [19]. cag8 (HP0528, cagX) is a component of T4SS (VirB9) encodes a membrane protein [19]. One strain lacked cagA gene but had other cagPAI genes indicating that cagA-positive isolates do not necessarily have to be cagPAI positive. Indian isolates had more Page 5/15 rearrangement in the cagPAI region compared to the Malay and the Chinese. Studies have shown that the subpopulations of H. pylori Indian isolates in our country consisted of mixed populations i.e., hpEurope, hpAsia2 and hpEAsia and this might re ect the diversity of cagPAI genes rearrangement among the Indian isolates [30,31]. The presence of speci c genes in H. pylori isolates associated with different ethnicities (cag4 in the Chinese isolates and cag2, cag14 and cag24 in the non-Chinese isolates) might represent strain associated disease outcomes. The cagA (VirB1) is a component of T4SS, whereas the function is still unknown for cag2, cag14 and cag24 [19]. Although the difference was not statistically signi cant, high frequency of cag2 was detected in gastric mucosa with CAG and IM/Atr and re ects the presence of this gene in non-Chinese isolates. These observations require further investigation to decipher the role of these genes. We found an association of cagPAI intactness with histopathological scores of the gastric mucosa. H. pylori harbouring partial cagPAI were associated with higher density of H. pylori and neutrophil activity, whereas H. pylori with deleted cagPAI causes increased in in ammatory score. The presence of neutrophil activity in the gastric mucosa is associated with CAG and this has been shown in our study that partial cagPAI H. pylori strains was detected more in CAG groups. As strains with deleted cagPAI only cause in ammation of the gastric mucosa, the presence of cagPAI proteins encoded by H. pylori strains is needed to cause more severe disease such as active gastritis and intestinal metaplasia. However, no speci c gene could be identi ed that causes severe condition. A group of genes encoded T4SS and for induction of IL-8 secretion have been shown to involve in the process of disease development [7,21]. Conclusions Results of the present study show that cagPAI organisation is diverse in isolates from different ethnicities. Comprehensive screening of the entire cagPAI genes provides a more accurate overview of the H. pylori cagPAI genotype and allows better identi cation of the virulence traits of the organisms in our multiracial population. H. pylori strains harbouring partial/rearrangement of the cagPAI genes associated with increased colonization and recruitment of neutrophil at the site of infection and further contribute to various disease outcomes caused by different genotypes of H. pylori strains. Methods Bacterial isolates A total of 95 non-repetitive H. pylori clinical isolates were obtained from patients (48 females and 47 males) recruited in the previous studies (research no. ETP-2013-042 and GUP-2011-307) between year 2011 to 2015. The patients’ population comprised of different ethnicities (15 Malays, 52 Chinese, 21 Indians and 7 others), with mean age of 53.71 ± 17.24 years old and age range from 17 to 83 years old. Biopsy samples from the antrum or corpus of the stomach from the patients were cultured for H. pylori Page 6/15 isolation. These isolates were then stored at -70C in brucella broth containing 15% glycerol. H. pylori were subcultured from frozen stock onto Columbia blood agar (Oxoid, Basingstoke, England) supplemented with 7% sheep blood and Dent’s supplement (Oxoid, Basingstoke, England) and incubated at 37°C for 5 to 7 days under microaerophilic environment. All patients had gastritis graded according to Updated Sydney Classi cation [18] except for two patients where the histopathological examination (HPE) results were not available. DNA extraction H. pylori colonies were scraped from the agar surface of Columbia blood agar plate and subjected to DNA extraction using FavorPrepTM Tissue Genomic DNA Extraction Mini kit according to the manufacturer’s instructions (Favorgen Biotech Corporation, Ping-Tung 908, Taiwan). DNA samples were diluted with ultrapure water to a concentration of 25 ng/µl and stored at -20°C until further processing. Determination of cagPAI genes The presence or absence of cagPAI in H. pylori strains was determined by PCR using primers for detection of the 5’ and 3’ anking region of the cagPAI as described by Olbermann et al., [19]. The ampli cations were carried out in 25 µl volume, each containing 12.5 µl mastermix (Lucigen, USA), 10 µl of each primers, 1 µl (25 ng) DNA and 10 µl DNAse and RNAse free sterile distilled water. PCR ampli cation for detection of cagPAI region consisted of initial denaturation at 95°C for 3 min, followed by 30 cycles of 95° for 30 s, 50°C for 60 s, and 72°C for 45 s, ending with nal extension at 72°C for 5 min. The ampli cations were performed in a PCR thermal cycler T100 Series (Bio-Rad, USA). The products were run on 1.5% agarose gel and stained with FloroSafe DNA stain (1st BASE Pte. Ltd, Singapore) and visualised with gel documentation (AlphaImager, Biosciences, CA). The cagPAI-positive isolates (n=91) were then subjected to subsequent PCRs for identi cation of all cagPAI genes using primers as described previously [19,20]. The deletion of HP0521 gene were con rmed using HP0521 empty site (ES) primer pair as described previously [21]. PCR ampli cation for cagPAI genes consisted of initial denaturation at 95°C for 3 min, followed by 30 cycles of 95° for 30s, annealing temperature for 60s (48C for cag11, 48.8C for cag3 and 55C for cag1, cag2, cag4, cag5, cag, cag6 to cag10, cag12 to cag26), and extension at 72°C for 45 s. A nal extension at 72°C for 5 min was performed for each PCR run. Representative positive PCR products (n=28) were sent for sequencing and the nucleotide sequences were blasted against NCBI databases to con rm the gene identity. Statistical analysis Statistical analysis was performed using SPSS software version 23 (SPSS Inc, Chicago, IL, USA). Differences between groups were evaluated using Chi-square (χ2) test, Yate’s continuity correction and Fisher’s exact probability test. Independent t-test was used to compared means between different groups Page 7/15 of histopathological scores. Score was represented with mean standard error of mean (SE). Differences were considered signi cant when p value was <0.05. Declarations Acknowledgments We would like to thank to the Universiti Kebangsaan Malaysia for providing both the permission and the facilities to conduct and publish this research and to the technical staffs of Dept. of Medical Microbiology & Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia for their technical help. Funding The research was funded by a grant from Ministry of Higher Education of Malaysia (grant no. FRGS/2/2014/SKK04/UKM/02/01). We also thank to Ministry of Higher Education of Malaysia for providing a studentship to SAR under the MyBrain15 program. Availability of data and materials Data will be shared upon request to the corresponding author al zah@ppukm.ukm.edu.my Authors’ contribution SAR performed all experiments and data analysis. HMN and NMZ participated in the study design and data analysis. AH involved in the design of the study, data analysis and manuscript writing. BSL participated in data analysis and manuscript writing. All authors read and approved the nal manuscript. Ethics approval and consent to participate The research protocol was approved by the Medical Research Ethic Committee of the university (UKM1.5.3.5/244/JEP-2016-095). The present study used H. pylori stock cultures where the informed consent was not applicable. However, these isolates were obtained from patients in previous studies (research no. ETP-2013-042 and GUP-2011-307) where informed consent was obtained from all the individuals included in the study. Competing interests The authors declare that they have no con ict of interest. 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Tables Table 1. Distribution of the cagPAI genes among 91 cagPAI-positive H. pylori isolates from patients with different ethnicities Page 11/15 Gene no. in 26695 strain Gene name Component of T4SS n (%) *Patients’ ethnicity, n (%) M (n=15) C (n=51) I (n=19) Other (n=6) HP0520 cag1 (cag) - 91 (100) 15 (100) 51 (100) 19 (100) 6 (100) HP521 cag2 - 32 (35.2) 13 (86.7) 5 (9.8) 11 (57.9) 3 (50) HP0522 cag3 (cag) u 90 (98.9) 15 (100) 51 (100) 18 (94.7) 6 (100) HP0523 cag4 (cag) VirB1 66 (72.5) 7 (46.7) 41 (80.4) 12 (63.2) 6 (100) HP0524 cag5 (cagβ) VirD4 90 (98.9) 15 (100) 51 (100) 19 (100) 5 (83.3) HP0525 cag VirB11 85 (93.4) 14 (93.3) 50 (98) 15 (78.9) 6 (100) HP0526 cag6 (cagZ) - 91 (100) 15 (100) 51 (100) 19 (100) 6 (100) HP0527 cag7 (cagY) VirB9 88 (96.7) 15 (100) 50 (98) 17 (89.5) 6 (100) HP0528 cag8 (cagX) VirB6 91 (100) 15 (100) 51 (100) 19 (100) 6 (100) HP0529 cag9 (cagW) VirB8 90 (98.9) 15 (100) 51 (100) 18 (94.7) 6 (100) HP0530 cag10 (cagV) - 88 (96.7) 14 (93.3) 50 (98) 18 (94.7) 6 (100) HP0531 cag11 (cagU) VirB7 77 (84.6) 15 (100) 41 (80.4) 15 (78.9) 6 (100) HP0532 cag12 (cagT) - 90 (98.9) 15 (100) 51 (100) 18 (94.7) 6 (100) HP0534 cag13 (cagS) - 89 (97.8) 15 (100) 51 (100) 17 (89.5) 6 (100) HP0535 cag14 (cagQ) - 48 (52.7) 14 (93.3) 20 (39.2) 10 (52.6) 4 (66.7) HP0536 cag15 (cagP) - 89 (97.8) 15 (100) 51 (100) 17 (89.5) 6 (100) HP0537 cag16 (cagM) u 84 (92.3) 15 (100) 49 (96.1) 14 (73.7) 6 (100) HP0538 cag17 - 86 15 (100) 48 17 6 Page 12/15 (cagN) (94.5) (94.1) (89.5) (100) HP0539 cag18 (cagL) VirB5 87 (95.6) 15 (100) 50 (98) 16 (84.2) 6 (100) HP0540 cag19 (cagI) - 83 (91.2) 15 (100) 48 (94.1) 16 (84.2) 4 (66.7) HP0541 cag20 (cagH) - 89 (97.8) 14 (93.3) 51 (100) 18 (94.7) 6 (100) HP0542 cag21 (cagG) - 91 (100) 15 (100) 51 (100) 19 (100) 6 (100) HP0543 cag22 (cagF) - 89 (95.6) 15 (100) 51 (100) 17 (89.5) 6 (100) HP0544 cag23 (cagE) VirB3/B4 87 (95.6) 15 (100) 50 (98) 16 (84.2) 6 (100) HP0545 cag24 (cagD) - 63 (69.2) 14 (93.3) 28 (54.9) 17 (89.5) 4 (66.7) HP0546 cag25 (cagC) VirB2 80 (87.9) 14 (93.3) 46 (90.2) 15 (78.9) 5 (83.3) HP0547 cag26 (cagA) effector 90 (98.9) 15 (100) 50 (98) 19 (100) 6 (100) M; Malays, C; Chinese, I; Indians, u; unknown function Table 2. Association of H. pylori cagPAI intactness with histopathological changes of gastric mucosa Page 13/15 Histopathological changes cagPAI, n (%) Score Intact Partial Deleted 0 7 (31.8) 15 (22.4) 1 (25) 1 10 (45.5) 21 (31.3) 2 (50) 2 4 (18.2) 18 (26.9) 1 (25) 3 1 (4.5) 13 (19.4) 0 Total score Mean SE 0.95 0.18 1.43 0.13 1.0 0.41 MNC in ltration2 0 0 1 (1.5) 0 1 6 (27.3) 23 (34.3) 0 2 14 (63.6) 36 (53.7) 4 (100) 3 2 (9.1) 7 (10.4) 0 Total score Mean SE 1.82 0.13 1.73 0.08 2.0 0 Neutrophil activity3 0 10 (45.5) 14 (20.9) 1 (25) 1 9 (40.9) 32 (47.8) 1 (25) 2 2 (9.1) 15 (22.4) 2 (50) 3 1 (4.5) 6 (9.0) 0 Total score Mean SE 0.73 0.18 1.19 0.11 1.25 0.48 Intestinal metaplasia 0 17 (77.3) 59 (88.1) 4 (100) 1 4 (18.2) 6 (9) 0 2 1 (4.5) 1 (1.5) 0 3 0 1 (1.5) 0 Total score Mean SE 0.27 0.12 0.16 0.06 0 Atrophy 0 15 (68.2) 53 (79.1) 3 (75) 1 5 (22.7) 10 (14.9) 1 (25) 2 1 (4.5) 2 (3) 0 3 1 (4.5) 2 (3) 0 Mean SE 0.45 0.17 0.30 0.08 0.25 0.25 H. pylori density1 Total score Page 14/15 Statistical analysis (Independent t-test): 1 Partial vs Intact; t = 2.166, p = 0.036, 95% CI (0.033-0.923) Partial vs Deleted; p = 0.42 Deleted vs Intact; p = 0.05 2 Deleted vs Partial; t = 3.308, p = 0.002, 95% CI (0.106 – 0.431) Deleted vs Intact; p = 0.162 Intact vs Partial; p = 0.586 3 Partial vs Intact; t = 2.20, p = 0.03, 95% CI (0.045 – 0.888) Deleted vs Intact; p = 0.266 Deleted vs Partial; p = 0.902 Table 3. cagPAI intactness in H. pylori in patients with different disease groups Disease group cagPAI, n (%) Intact (n=22) Partial (n=66) Deleted (n=4) CG 7 (3.8) 12 (18.2) 1 (25) CAG 6 (13.6) 36 (54.5) 2 (50) IM/Atr 9 (40.9) 18 (27.3) 1 (25) Intact vs partial: 2 = 4.992, df = 2, p = 0.08 Supplementary Files This is a list of supplementary les associated with this preprint. 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