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Helicobacter heilmannii Colonization Is Associated with High Risk for Gastritis
Archives of Medical Research 50 (2019) 423e427
ORIGINAL ARTICLE
Helicobacter heilmannii Colonization Is Associated With High Risk for Gastritis Mahnaz Mohammadi,a Amin Talebi Bezmin Abadi,a Farid Rahimi,b and Mojgan Forootanc a
Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran b Research School of Biology, The Australian National University, Canberra, Australia c Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran Received for publication September 9, 2019; accepted November 7, 2019 (ARCMED_2019_812).
Introduction. We aimed to study potential associations between colonization by four common non-pylori Helicobacter species and gastroduodenal diseases by comparing samples from patients infected with H. pylori with samples from non-infected subjects. Materials and Methods. Patients (n 5 190) who were subjected to upper gastrointestinal endoscopy because of gastroduodenal conditions were enrolled in this cross-sectional study. Antral biopsy samples were taken from patients in two major hospitals (Mehrad and Imam-Hossein) in Tehran, Iran, during 2017e2018. DNA was isolated from the biopsy specimens, and PCR amplification was used to identify the Helicobacter species by using their corresponding specific primer sets. Results. Out of 120 cases positive for H. pylori, 46 (38%) were patients with gastritis, 23 (19%) with duodenal ulcer, 11 (9%) with gastric cancer, and 40 (33.3%) with gastric ulcer. Overall, 70 (36%) patients were negative for H. pylori. H. pylori cases were uninfected by any of the other tested Helicobacter species. Among the 70 patients without H. pylori, 34 had gastritis—31 (94%) of these were positive also for H. heilmannii ( p 5 0.001, Odds Ratio: 51.6; 95% Confidence Intervals: 11.8e225.6). We did not find any patient carrying mixed Helicobacter infections with any non-pylori Helicobacter species in this cohort. Conclusions. Given our evidence about the possibility of involvement of H. heilmannii in patients suffering from gastritis and nonexistence of mixed non-pylori Helicobacter infections, bacteriological testing of subjects negative for H. pylori becomes clinically relevant and important. Ó 2019 IMSS. Published by Elsevier Inc. Key Words: Gastritis, Helicobacter heilmannii, Helicobacter pylori, Non-pylori Helicobacter species, PCR.
Introduction Helicobacter pylori has long been known as a colonizer of gastric mucosa in at least 50% of the population globally (1). A significant link exists between H. pylori infection and development and progression of severe digestive disorders. These disorders include chronic gastritis and peptic ulcer among at least 80% of individuals, and gastric cancer Address reprint requests to: Amin Talebi Bezmin Abadi, PhD, Assistant Professor, Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-111, Tehran, Iran; Phone: (þ98) (21) 8288 4883 or (þ98) (21) 8288 4803; E-mail: Amin.talebi@ modares.ac.ir
at least in 2e5% of colonized individuals (2e4). Nevertheless, H. pylori is not the only gastric colonizer of Helicobacter species (5e7). Some other clinical gastroduodenal conditions reportedly have been associated with Helicobacter species other than H. pylori; these species include H. suis, H. canis, H. bizzozeronii, and H. heilmannii (8e11). The latter species have been postulated to underlie the pathogenesis of gastritis or duodenal ulcers in humans (12e16). Yamamoto K, et al. were first to describe the ability of H. suis to colonize the human stomach and cause lifelong clinical outcomes if left untreated (17). Accordingly, H. sius has been one of the most frequent non-pylori Helicobacter species isolated from the human stomach samples
0188-4409/$ - see front matter. Copyright Ó 2019 IMSS. Published by Elsevier Inc. https://doi.org/10.1016/j.arcmed.2019.11.001
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(in 10e29% of cases). Weight loss and gastritis were two major signs described in hosts infected with H. sius (18). In 1993, Burnens AP, et al. described a Campylobacter-like organism, resembling Helicobacter fennelliae; later this organism was proven to be H. canis because it was mainly isolated from canine feces (19). However, further investigations, including 16s rRNA sequencing, showed that humans also can be colonized by this zoonotic microorganism. Lastly, H. heilmannii was found in !5% of symptomatic patients; however, differences in colonization by H. heilmannii between pylori-negative individuals and pylori-positive patients have not been studied (11,12,20). We hypothesized that colonization with H. pylori may be accompanied with colonization by some other Helicobacter species. Iran with a population of 80 million presently shows rising prevalence of H. pylori and non-pylori Helicobacter infections (21e23). However, accurate data on clinical outcomes of colonization by Helicobacter species in Iran are lacking. Therefore, here, we aimed to ascertain whether an association between colonization by four common nonpylori Helicobacter species exists in pylori-negative subjects and patients infected with H. pylori. We used antral biopsy samples taken from the above subjects who were referred to two main hospitals (Mehrad and ImamHossein) in Tehran, Iran, during 2017e2018.
Materials and Methods Patients’ Samples Individuals (n 5 190; mean age 5 52 (range, 21e69); 48% female), who had gastroduodenal complaints and were subjected to upper gastrointestinal endoscopy, were enrolled. Participation in the study was voluntary. The participants signed informed consent forms. The study protocols were approved by the Clinical Research Ethics Committee of Tarbiat Modares University, Tehran, Iran (IR.MODARES.REC.1397.241). Subjects’ antral biopsy samples were obtained by gastric endoscopy performed by trained surgeons. Subjects were categorized as pylori-positive or pylori-negative based on PCR experiments assaying for a pylori-specific housekeeping gene, glmM. We documented the subjects’ demographic and clinical characteristics by conducting interviews. The study’s exclusion criteria included history of gastric surgery; previous treatment with non-steroidal anti-inflammatory drugs, proton-pump inhibitors, or antibiotics against H. pylori three months before endoscopy; and being under 18 or older than 75 years of age. PCR Amplification Genomic DNA was extracted from the study subjects’ antral biopsy samples by using a tissue DNA-extraction
kit (QIAGEN, Germany) with minor modifications. Extracted DNA was stored at 20 C until genotyping was performed. For detecting the four non-pylori Helicobacter species, we used the corresponding primer sets as published previously (14,24e26). glmM-specific primer sets were used according to published protocols to confirm H. pylori infections (27). Briefly, amplification was performed in 20 mL PCR mixtures—containing 1 mL of extracted genomic DNA (approximately 5e10 ng), 0.75 mM of each primer set corresponding to the tested Helicobacter species (synthesized by Takapozist Company, Tehran, Iran), 1.5 mL of dNTPs at 0.2 mM, and 8 mL of 2 master mix (Ampliqon, Copenhagen, Denmark)—and by using a T100 Thermal Cycler (Bio-Rad, Berkeley, California). All PCR products were visualized by electrophoresis on 1.5% agarose gels (Sina-clon, Tehran, Iran) which were then stained with GelRedÒ and subjected to UV transillumination (Biometra, Germany) and imaging. We included positive controls obtained from previously diagnosed clinical samples and negative controls (excluding DNA template) to ensure experimental quality. Statistical Analyses A c2 test was used to calculate the association between the presence of four non-pylori Helicobacter species. A p-value of !0.05 was deemed to be statistically significant.
Results Gender and age distribution of pylori-positive and pylorinegative patients were not significantly different. Of 190 subjects, 63% were pylori-positive and 36% pylori-negative according to our PCR experiments assaying for glmM. Of 120 pylori-positive cases, 46 had gastritis, 23 had duodenal ulcer, 11 gastric cancer, and 40 gastric ulcer. None of the pylori-positive patients harbored the other tested non-pylori Helicobacter species. Prevalence of Non-pylori Helicobacter Species Of the 70 pylori-negative cases, 34 (49%) patients were diagnosed with gastritis, 30 (43%) with duodenal ulcer, and six (8%) with gastric cancer. Among these, 38 (54%) were infected with H. heilmannii, three were infected with H. bizzozeronii, two with gastritis, and one with duodenal ulcer. Of the 34 patients with gastritis, 31 (91%) carried H. heilmannii ( p 5 0.001, odds ratio: 51.6; 95% confidence interval: 11.8e225.6). We had only one patient harboring H. bizzozeronii (3%) in the duodenal ulcer group. Out of 30 patients diagnosed with duodenal ulcer, six were positive for H. heilmannii and one for H. bizzozeronii. We also found two patients with chronic gastritis carrying H. bizzozeronii (Table 1). The prevalence of H. heilmannii was significant among the patients with duodenal ulcer ( p !0.001,
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Table 1. Distribution of the four non-pylori Helicobacter species among the 70 pylori-negative patients Non-pylori Helicobacter species Diagnoses
H. heilmannii
H. bizzozeronii
Gastritis (n 5 34) Duodenal ulcer (n 5 30) Gastric cancer (n 5 6)
31 (91%) ( p 5 0.001, OR: 51.6; 95% CI: 11.8e225.6) 6 (20%) ( p !0.001, OR: 0.07; 95% CI: 0.02e0.2) 1 (16%) ( p 5 0.09, OR: 0.1; 95% CI: 0.01e1.4)
2 (5%) ( p 5 0.6, Odds Ratio: 2.1; 95% CI: 0.2e22.3) 1 (3%) ( p 5 0.6, OR: 1.3; 95% CI: 0.08e22.4) 0
OR, odds ratio; CI, confidence interval.
OR: 0.07; 95% CI: 0.02e0.2). We did not find any patient carrying mixed infections with any of the tested non-pylori Helicobacter species.
Discussion Scientists had long believed that the human gastric environment is germ-free, and that no living microorganism could survive the harsh acidic environment of the stomach. The groundbreaking discovery by Marshal and Warren in 1983 proved that H. pylori was alive in the human stomach (28). Subsequently, the belief that H. pylori is the only microorganism that can survive in the gastric environment continued for another two decades; however, some contemporary studies have shown gastric colonization by some non-pylori Helicobacter species (29). Accordingly, researchers now strive to understand any potential associations between colonization by non-pylori Helicobacter species and causation of morbid gastroduodenal conditions, including duodenal ulcer, gastritis, and gastric cancer (30,31). According to a 2012 report by Smet A, et al., H. heilmannii was successfully isolated from feline gastric mucosa; however, the clinical importance of this gastric infection is still not fully appreciated in humans (32). Before this study, Andersen LP, et al. had confirmed culturing an organism akin to H. heilmannii from a symptomatic patient (33). This was later confirmed by a Finish group to be colonization by H. bizzozeronii in patients with severe dyspeptic symptoms (25). McNulty CA, et al. were the first to report that this spiral bacterium, which was provisionally called Gastrospirillum hominis, could be isolated from the human gastric mucosa (34). Despite these discoveries, the challenge of clarifying disease causation by non-pylori Helicobacter species still continues (35e37). The rationale for our research was to shed light on the causation associations with Helicobacter species considering a background of conflicting reports published in the later few decades. Here we enrolled a moderate cohort of subjects to investigate such potential associations, aiming to better understand the non-pylori Helicobacter species. We tested for colonization by four non-pylori Helicobacter species, including H. heilmannii, H. canis, H. suis, and H. bizzozeronii, using the antral biopsy samples obtained from
individuals with gastroduodenal conditions. We found no difference in age and gender among subjects with any of the four non-pylori Helicobacter species and those with H. pylori infections. H. heilmannii was the most prevalent non-pylori Helicobacter species among the subjects with gastritis ( p 5 0.001, OR: 51.6; 95% CI: 11.8e225.6). Among the 34 patients with gastritis and 30 patients with duodenal ulcer, we found one subject infected with H. bizzozeronii in each group. Similar to the report by Van den Bulck K, et al., (31) the prevalence of H. bizzozeronii (3%) among the 30 patients with duodenal ulcer was similar to that in gastritis patients (5%). We detected six cases with gastric cancer who presented one positive sample for H. heilmannii ( p 5 0.09, OR: 0.1; 95% CI: 0.01e1.4). The low number of cases with gastritis did not allow for a significant statistical analysis; however, we aim to study a larger high-risk group of the patients in the future. Interestingly, of the 30 patients with duodenal ulcer, 20% were positive for H. heilmannii ( p !0.001, OR: 0.07; 95% CI: 0.02e0.2). To the best of our knowledge, ours is the first crosssectional study representing an Iranian population sample investigated for the clinical relevance of the four tested non-pylori Helicobacter species. Importantly, we found no mixed Helicobacter infections although 5% of H. pylori isolates reportedly represent mixed infections (38); thus, some of the tested non-pylori Helicobacter species are expected to be present in mixed microniches. Notably, we found no biopsy samples positive for H. suis or H. canis. Unsurprisingly, this result is consistent with some previous reports (24,39,40). We cannot exclude the possibility of finding higher rates of infection if populations sampling cat owners or dog owners were studied; this is potentially one limitation of our study. Our study is also limited by sample size. Further cross-sectional studies of larger sample sizes are needed to establish a clear causative association between H. heilmannii colonization and gastritis. We conclude that infections with non-pylori Helicobacter species are candidates for further microbiological testing for targeted and improved clinical management. The zoonotic nature of some of these species necessitates deliberation about pet ownership. Accordingly, examining the veterinary subjects for prevalence of non-pylori Helicobacter infections will be crucial and informative. Testing for H. heilmannii in suspect gastritis patients with non-
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pylori Helicobacter species must be considered clinically when managing such patients. Many gastroenterologists may administer differently targeted treatment regimens if they do not find H. pylori positivity. Therefore, screening for H. heilmannii in suspect gastritis patients with non-pylori Helicobacter species can be a novel, but potentially important, approach in the management of such individuals.
Acknowledgment The present study was financially supported by the grant 94014027 by the Iran National Science Foundation (INSF).
Conflict of Interest None.
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15. Ierardi E, Monno RA, Gentile A, et al. Helicobacter heilmannii gastritis: a histological and immunohistochemical trait. J Clin Pathol 2001;54:774e777. 16. Jhala D, Jhala N, Lechago J, et al. Helicobacter heilmannii gastritis: association with acid peptic diseases and comparison with Helicobacter pylori gastritis. Mod Pathol 1999;12:534e538. 17. Yamamoto K, Nishiumi S, Yang L, et al. Anti-CXCL13 antibody can inhibit the formation of gastric lymphoid follicles induced by Helicobacter infection. Mucosal Immunol 2014;7:1244e1254. 18. Flahou B, Haesebrouck F, Pasmans F, et al. Helicobacter suis causes severe gastric pathology in mouse and mongolian gerbil models of human gastric disease. PLoS One 2010;5:e14083. 19. Burnens AP, Stanley J, Schaad UB, et al. Novel Campylobacter-like organism resembling Helicobacter fennelliae isolated from a boy with gastroenteritis and from dogs. J Clin Microbiol 1993;31: 1916e1917. 20. Nakamura M, Øverby A, Murayama SY, et al. Gastric non-Helicobacter pylori Helicobacter: its significance in human gastric diseases. Helicobacter pylori. Springer, 2016131e140. 21. Kalantar E, Gharavi MJ, Oshaghi M, et al. Seroprevalence of Helicobacter pylori infection in Iranian adolescents: the CASPIAN-III study. Int J Pediatr 2017;5:4251e4256. 22. Moosazadeh M, Lankarani KB, Afshari M. Meta-analysis of the prevalence of Helicobacter pylori infection among children and adults of Iran. Int J Prev Med 2016;7:48. 23. Yadegar A, Alebouyeh M, Lawson AJ, et al. Differentiation of nonpylori Helicobacter species based on PCRerestriction fragment length polymorphism of the 23S rRNA gene. World J Microbiol Biotechnol 2014;30:1909e1917. 24. Baele M, Decostere A, Vandamme P, et al. Isolation and characterization of Helicobacter suis sp. nov. from pig stomachs. Int J Syst Evol Microbiol 2008;58:1350e1358. 25. Kivist€o R, Linros J, Rossi M, et al. Characterization of multiple Helicobacter bizzozeronii isolates from a Finnish patient with severe dyspeptic symptoms and chronic active gastritis. Helicobacter 2010;15:58e66. 26. Tankovic J, Smati M, Lamarque D, et al. First detection of Helicobacter canis in chronic duodenal ulcerations from a patient with Crohn’s disease. Inflamm Bowel Dis 2011;17:1830e1831. 27. Chomvarin C, Namwat W, Chaicumpar K, et al. Prevalence of Helicobacter pylori vacA, cagA, cagE, iceA and babA2 genotypes in Thai dyspeptic patients. Int J Infect Dis 2008;12:30e36. 28. Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984;1: 1311e1315. 29. O’Rourke JL, Grehan M, Lee A. Non-pylori Helicobacter species in humans. Gut 2001;49:601e606. 30. Nishikawa K, Sugiyama T, Kato M, et al. Non-Helicobacter pylori and non-NSAID peptic ulcer disease in the Japanese population. Eur J Gastroenterol Hepatol 2000;12:635e640. 31. Van den Bulck K, Decostere A, Baele M, et al. Identification of nonHelicobacter pylori spiral organisms in gastric samples from humans, dogs, and cats. J Clin Microbiol 2005;43:2256e2260. 32. Smet A, Flahou B, D’Herde K, et al. Helicobacter heilmannii sp. nov., isolated from feline gastric mucosa. Int J Syst Evol Microbiol 2012;62:299e306. 33. Andersen LP, Norgaard A, Holck S, et al. Isolation of a ‘‘Helicobacter heilmanii’’-like organism from the human stomach. Eur J Clin Microbiol Infect Dis 1996;15:95e96. 34. McNulty CA, Dent JC, Curry A, et al. New spiral bacterium in gastric mucosa. J Clin Pathol 1989;42:585e591. 35. Figura N, Guglielmetti P, Quaranta S. Spiral shaped bacteria in gastric mucosa. J Clin Pathol 1990;43:173. 36. Bento-Miranda M, Figueiredo C. Helicobacter heilmannii sensu lato: an overview of the infection in humans. World J Gastroenterol 2014; 20:17779e17787.
Helicobacter Heilmannii in Gastritis Patients 37. Trebesius K, Adler K, Vieth M, et al. Specific detection and prevalence of Helicobacter heilmannii-like organisms in the human gastric mucosa by fluorescent in situ hybridization and partial 16S ribosomal DNA sequencing. J Clin Microbiol 2001;39: 1510e1516. 38. Kusters JG, van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 2006;19:449e490.
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39. Hellemans A, Chiers K, De Bock M, et al. Prevalence of ’Candidatus Helicobacter suis’ in pigs of different ages. Vet Rec 2007;161: 189e192. 40. Grasso GM, Ripabelli G, Sammarco ML, et al. Prevalence of Helicobacter-like organisms in porcine gastric mucosa: a study of swine slaughtered in Italy. Comp Immunol Microbiol Infect Dis 1996;19: 213e217.
ORIGINAL ARTICLE
Helicobacter heilmannii Colonization Is Associated With High Risk for Gastritis Mahnaz Mohammadi,a Amin Talebi Bezmin Abadi,a Farid Rahimi,b and Mojgan Forootanc a
Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran b Research School of Biology, The Australian National University, Canberra, Australia c Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran Received for publication September 9, 2019; accepted November 7, 2019 (ARCMED_2019_812).
Introduction. We aimed to study potential associations between colonization by four common non-pylori Helicobacter species and gastroduodenal diseases by comparing samples from patients infected with H. pylori with samples from non-infected subjects. Materials and Methods. Patients (n 5 190) who were subjected to upper gastrointestinal endoscopy because of gastroduodenal conditions were enrolled in this cross-sectional study. Antral biopsy samples were taken from patients in two major hospitals (Mehrad and Imam-Hossein) in Tehran, Iran, during 2017e2018. DNA was isolated from the biopsy specimens, and PCR amplification was used to identify the Helicobacter species by using their corresponding specific primer sets. Results. Out of 120 cases positive for H. pylori, 46 (38%) were patients with gastritis, 23 (19%) with duodenal ulcer, 11 (9%) with gastric cancer, and 40 (33.3%) with gastric ulcer. Overall, 70 (36%) patients were negative for H. pylori. H. pylori cases were uninfected by any of the other tested Helicobacter species. Among the 70 patients without H. pylori, 34 had gastritis—31 (94%) of these were positive also for H. heilmannii ( p 5 0.001, Odds Ratio: 51.6; 95% Confidence Intervals: 11.8e225.6). We did not find any patient carrying mixed Helicobacter infections with any non-pylori Helicobacter species in this cohort. Conclusions. Given our evidence about the possibility of involvement of H. heilmannii in patients suffering from gastritis and nonexistence of mixed non-pylori Helicobacter infections, bacteriological testing of subjects negative for H. pylori becomes clinically relevant and important. Ó 2019 IMSS. Published by Elsevier Inc. Key Words: Gastritis, Helicobacter heilmannii, Helicobacter pylori, Non-pylori Helicobacter species, PCR.
Introduction Helicobacter pylori has long been known as a colonizer of gastric mucosa in at least 50% of the population globally (1). A significant link exists between H. pylori infection and development and progression of severe digestive disorders. These disorders include chronic gastritis and peptic ulcer among at least 80% of individuals, and gastric cancer Address reprint requests to: Amin Talebi Bezmin Abadi, PhD, Assistant Professor, Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-111, Tehran, Iran; Phone: (þ98) (21) 8288 4883 or (þ98) (21) 8288 4803; E-mail: Amin.talebi@ modares.ac.ir
at least in 2e5% of colonized individuals (2e4). Nevertheless, H. pylori is not the only gastric colonizer of Helicobacter species (5e7). Some other clinical gastroduodenal conditions reportedly have been associated with Helicobacter species other than H. pylori; these species include H. suis, H. canis, H. bizzozeronii, and H. heilmannii (8e11). The latter species have been postulated to underlie the pathogenesis of gastritis or duodenal ulcers in humans (12e16). Yamamoto K, et al. were first to describe the ability of H. suis to colonize the human stomach and cause lifelong clinical outcomes if left untreated (17). Accordingly, H. sius has been one of the most frequent non-pylori Helicobacter species isolated from the human stomach samples
0188-4409/$ - see front matter. Copyright Ó 2019 IMSS. Published by Elsevier Inc. https://doi.org/10.1016/j.arcmed.2019.11.001
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(in 10e29% of cases). Weight loss and gastritis were two major signs described in hosts infected with H. sius (18). In 1993, Burnens AP, et al. described a Campylobacter-like organism, resembling Helicobacter fennelliae; later this organism was proven to be H. canis because it was mainly isolated from canine feces (19). However, further investigations, including 16s rRNA sequencing, showed that humans also can be colonized by this zoonotic microorganism. Lastly, H. heilmannii was found in !5% of symptomatic patients; however, differences in colonization by H. heilmannii between pylori-negative individuals and pylori-positive patients have not been studied (11,12,20). We hypothesized that colonization with H. pylori may be accompanied with colonization by some other Helicobacter species. Iran with a population of 80 million presently shows rising prevalence of H. pylori and non-pylori Helicobacter infections (21e23). However, accurate data on clinical outcomes of colonization by Helicobacter species in Iran are lacking. Therefore, here, we aimed to ascertain whether an association between colonization by four common nonpylori Helicobacter species exists in pylori-negative subjects and patients infected with H. pylori. We used antral biopsy samples taken from the above subjects who were referred to two main hospitals (Mehrad and ImamHossein) in Tehran, Iran, during 2017e2018.
Materials and Methods Patients’ Samples Individuals (n 5 190; mean age 5 52 (range, 21e69); 48% female), who had gastroduodenal complaints and were subjected to upper gastrointestinal endoscopy, were enrolled. Participation in the study was voluntary. The participants signed informed consent forms. The study protocols were approved by the Clinical Research Ethics Committee of Tarbiat Modares University, Tehran, Iran (IR.MODARES.REC.1397.241). Subjects’ antral biopsy samples were obtained by gastric endoscopy performed by trained surgeons. Subjects were categorized as pylori-positive or pylori-negative based on PCR experiments assaying for a pylori-specific housekeeping gene, glmM. We documented the subjects’ demographic and clinical characteristics by conducting interviews. The study’s exclusion criteria included history of gastric surgery; previous treatment with non-steroidal anti-inflammatory drugs, proton-pump inhibitors, or antibiotics against H. pylori three months before endoscopy; and being under 18 or older than 75 years of age. PCR Amplification Genomic DNA was extracted from the study subjects’ antral biopsy samples by using a tissue DNA-extraction
kit (QIAGEN, Germany) with minor modifications. Extracted DNA was stored at 20 C until genotyping was performed. For detecting the four non-pylori Helicobacter species, we used the corresponding primer sets as published previously (14,24e26). glmM-specific primer sets were used according to published protocols to confirm H. pylori infections (27). Briefly, amplification was performed in 20 mL PCR mixtures—containing 1 mL of extracted genomic DNA (approximately 5e10 ng), 0.75 mM of each primer set corresponding to the tested Helicobacter species (synthesized by Takapozist Company, Tehran, Iran), 1.5 mL of dNTPs at 0.2 mM, and 8 mL of 2 master mix (Ampliqon, Copenhagen, Denmark)—and by using a T100 Thermal Cycler (Bio-Rad, Berkeley, California). All PCR products were visualized by electrophoresis on 1.5% agarose gels (Sina-clon, Tehran, Iran) which were then stained with GelRedÒ and subjected to UV transillumination (Biometra, Germany) and imaging. We included positive controls obtained from previously diagnosed clinical samples and negative controls (excluding DNA template) to ensure experimental quality. Statistical Analyses A c2 test was used to calculate the association between the presence of four non-pylori Helicobacter species. A p-value of !0.05 was deemed to be statistically significant.
Results Gender and age distribution of pylori-positive and pylorinegative patients were not significantly different. Of 190 subjects, 63% were pylori-positive and 36% pylori-negative according to our PCR experiments assaying for glmM. Of 120 pylori-positive cases, 46 had gastritis, 23 had duodenal ulcer, 11 gastric cancer, and 40 gastric ulcer. None of the pylori-positive patients harbored the other tested non-pylori Helicobacter species. Prevalence of Non-pylori Helicobacter Species Of the 70 pylori-negative cases, 34 (49%) patients were diagnosed with gastritis, 30 (43%) with duodenal ulcer, and six (8%) with gastric cancer. Among these, 38 (54%) were infected with H. heilmannii, three were infected with H. bizzozeronii, two with gastritis, and one with duodenal ulcer. Of the 34 patients with gastritis, 31 (91%) carried H. heilmannii ( p 5 0.001, odds ratio: 51.6; 95% confidence interval: 11.8e225.6). We had only one patient harboring H. bizzozeronii (3%) in the duodenal ulcer group. Out of 30 patients diagnosed with duodenal ulcer, six were positive for H. heilmannii and one for H. bizzozeronii. We also found two patients with chronic gastritis carrying H. bizzozeronii (Table 1). The prevalence of H. heilmannii was significant among the patients with duodenal ulcer ( p !0.001,
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Table 1. Distribution of the four non-pylori Helicobacter species among the 70 pylori-negative patients Non-pylori Helicobacter species Diagnoses
H. heilmannii
H. bizzozeronii
Gastritis (n 5 34) Duodenal ulcer (n 5 30) Gastric cancer (n 5 6)
31 (91%) ( p 5 0.001, OR: 51.6; 95% CI: 11.8e225.6) 6 (20%) ( p !0.001, OR: 0.07; 95% CI: 0.02e0.2) 1 (16%) ( p 5 0.09, OR: 0.1; 95% CI: 0.01e1.4)
2 (5%) ( p 5 0.6, Odds Ratio: 2.1; 95% CI: 0.2e22.3) 1 (3%) ( p 5 0.6, OR: 1.3; 95% CI: 0.08e22.4) 0
OR, odds ratio; CI, confidence interval.
OR: 0.07; 95% CI: 0.02e0.2). We did not find any patient carrying mixed infections with any of the tested non-pylori Helicobacter species.
Discussion Scientists had long believed that the human gastric environment is germ-free, and that no living microorganism could survive the harsh acidic environment of the stomach. The groundbreaking discovery by Marshal and Warren in 1983 proved that H. pylori was alive in the human stomach (28). Subsequently, the belief that H. pylori is the only microorganism that can survive in the gastric environment continued for another two decades; however, some contemporary studies have shown gastric colonization by some non-pylori Helicobacter species (29). Accordingly, researchers now strive to understand any potential associations between colonization by non-pylori Helicobacter species and causation of morbid gastroduodenal conditions, including duodenal ulcer, gastritis, and gastric cancer (30,31). According to a 2012 report by Smet A, et al., H. heilmannii was successfully isolated from feline gastric mucosa; however, the clinical importance of this gastric infection is still not fully appreciated in humans (32). Before this study, Andersen LP, et al. had confirmed culturing an organism akin to H. heilmannii from a symptomatic patient (33). This was later confirmed by a Finish group to be colonization by H. bizzozeronii in patients with severe dyspeptic symptoms (25). McNulty CA, et al. were the first to report that this spiral bacterium, which was provisionally called Gastrospirillum hominis, could be isolated from the human gastric mucosa (34). Despite these discoveries, the challenge of clarifying disease causation by non-pylori Helicobacter species still continues (35e37). The rationale for our research was to shed light on the causation associations with Helicobacter species considering a background of conflicting reports published in the later few decades. Here we enrolled a moderate cohort of subjects to investigate such potential associations, aiming to better understand the non-pylori Helicobacter species. We tested for colonization by four non-pylori Helicobacter species, including H. heilmannii, H. canis, H. suis, and H. bizzozeronii, using the antral biopsy samples obtained from
individuals with gastroduodenal conditions. We found no difference in age and gender among subjects with any of the four non-pylori Helicobacter species and those with H. pylori infections. H. heilmannii was the most prevalent non-pylori Helicobacter species among the subjects with gastritis ( p 5 0.001, OR: 51.6; 95% CI: 11.8e225.6). Among the 34 patients with gastritis and 30 patients with duodenal ulcer, we found one subject infected with H. bizzozeronii in each group. Similar to the report by Van den Bulck K, et al., (31) the prevalence of H. bizzozeronii (3%) among the 30 patients with duodenal ulcer was similar to that in gastritis patients (5%). We detected six cases with gastric cancer who presented one positive sample for H. heilmannii ( p 5 0.09, OR: 0.1; 95% CI: 0.01e1.4). The low number of cases with gastritis did not allow for a significant statistical analysis; however, we aim to study a larger high-risk group of the patients in the future. Interestingly, of the 30 patients with duodenal ulcer, 20% were positive for H. heilmannii ( p !0.001, OR: 0.07; 95% CI: 0.02e0.2). To the best of our knowledge, ours is the first crosssectional study representing an Iranian population sample investigated for the clinical relevance of the four tested non-pylori Helicobacter species. Importantly, we found no mixed Helicobacter infections although 5% of H. pylori isolates reportedly represent mixed infections (38); thus, some of the tested non-pylori Helicobacter species are expected to be present in mixed microniches. Notably, we found no biopsy samples positive for H. suis or H. canis. Unsurprisingly, this result is consistent with some previous reports (24,39,40). We cannot exclude the possibility of finding higher rates of infection if populations sampling cat owners or dog owners were studied; this is potentially one limitation of our study. Our study is also limited by sample size. Further cross-sectional studies of larger sample sizes are needed to establish a clear causative association between H. heilmannii colonization and gastritis. We conclude that infections with non-pylori Helicobacter species are candidates for further microbiological testing for targeted and improved clinical management. The zoonotic nature of some of these species necessitates deliberation about pet ownership. Accordingly, examining the veterinary subjects for prevalence of non-pylori Helicobacter infections will be crucial and informative. Testing for H. heilmannii in suspect gastritis patients with non-
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pylori Helicobacter species must be considered clinically when managing such patients. Many gastroenterologists may administer differently targeted treatment regimens if they do not find H. pylori positivity. Therefore, screening for H. heilmannii in suspect gastritis patients with non-pylori Helicobacter species can be a novel, but potentially important, approach in the management of such individuals.
Acknowledgment The present study was financially supported by the grant 94014027 by the Iran National Science Foundation (INSF).
Conflict of Interest None.
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