- Email: [email protected]
The cagA status of Helicobacter pylori isolates from dyspeptic children in Turkey
FEMS Immunology and Medical Microbiology 36 (2003) 147^149
www.fems-microbiology.org
The cagA status of Helicobacter pylori isolates from dyspeptic children in Turkey Inci Nur Salt|k a b
a;
º zge Darka Ertunc# b , , Hu«lya Demir a , Doruk Engin b , O Yakut Akyo«n b , Nurten Koc#ak a
Department of Pediatrics, Section of Gastroenterology, Hacettepe University, Faculty of Medicine, 06100 Ankara, Turkey Department of Clinical Microbiology and Microbiology, Hacettepe University, Faculty of Medicine, 06100 Ankara, Turkey Received 4 July 2002; received in revised form 16 October 2002; accepted 4 November 2002 First published online 6 February 2003
Abstract There are inconsistent reports regarding cytotoxin-associated gene A (cagA) status of Helicobacter pylori isolates and the severity of the mucosal lesions in children. The aim of this study was to determine the prevalence of cagAþ strains and to evaluate its correlation with clinic and endoscopic findings. We examined 45 H. pylori strains that were grown on brain^heart infusion agar supplemented with 7% horse blood. Following 72 h of incubation colonies were harvested and bacterial DNA was extracted. Polymerase chain reaction primers F1 and B1 were used to amplify a 348-bp internal fragment of cagA. The prevalence of cagA in Turkish pediatric patients was 55.6%. No association was found between cagA status and the severity of gastro-duodenal lesions. @ 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Helicobacter pylori; cagA; Antral nodularity; Children
1. Introduction Helicobacter pylori infection is usually acquired during childhood and in most children its presence does not lead to clinically apparent disease, even when the organism colonizing the gastric mucosa causes chronic active gastritis. This organism is associated with a signi¢cant proportion of duodenal ulcers and there are epidemiologic data linking chronic H. pylori infection with the development of gastric adenocarcinoma and gastric lymphoma [1,2]. It remains unclear why a minority of H. pylori positive patients develop the disease. It has been suggested that H. pylori strains containing the cytotoxin-associated gene A (cagA) pathogenicity island are more virulent and are associated with the complication of the infection, such as peptic ulcers. The cagA contains a gene that encodes a highly immunogenic outer-membrane protein with a molecular mass of 120^140 kDa and seems to induce an increased in£ammation in the gastric mucosa through release of in-
* Corresponding author. Tel. : +90 (312) 305 1993 and 1994; Fax : +90 (312) 311 7715. E-mail address : [email protected] (I.N. Salt|k).
terleukin-8 (IL-8) by epithelial cells [3]. Results of studies performed in children showed that cagA-positive (cagAþ ) strains of H. pylori caused more severe gastric in£ammation and lower susceptibility to eradication treatment [4^6]. However, in Japanese children, although the prevalence of cagAþ H. pylori strains was high, there was no association with nodular gastritis or peptic ulcer disease [7]. Thus, inconsistent reports exist regarding cagA status of H. pylori isolates and the severity of the mucosal lesions in children. The aim of this study was to determine the prevalence of cagAþ strains in H. pylori isolates from symptomatic children and to evaluate its correlation with clinic and endoscopic ¢ndings.
2. Materials and methods 2.1. Patients H. pylori strains were isolated from 45 patients (22 male ; 48.9%) aged between 6^16 years (mean K S.D.: 11.1 K 2.5 years). Thirty-nine (86.7%) patients had complaints of abdominal pain as the main problem. Other
0928-8244 / 03 / $22.00 @ 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0928-8244(03)00024-5
FEMSIM 1502 23-4-03
148
I.N. Salt|k et al. / FEMS Immunology and Medical Microbiology 36 (2003) 147^149
complaints were halitosis, growth retardation, vomiting and nausea. The mean duration of the symptoms was 15.8 ( K 23.2) months. 2.2. Isolation of H. pylori After informed consent was obtained, an esophagogastroduodenoscopy (EGD) was performed with an Olympus GIF P100 endoscope. Intravenous sedation with midazolam and pethidine was administered to all children before endoscopic examination. Endoscopic ¢ndings were noted for the presence or absence of abnormalities such as antral nodularity, ulcers or erythema. At least four antral gastric biopsies were obtained from each patient for histology, culture and rapid urease test medium (Dio-Helico, Diomed). H. pylori was recovered from one antral biopsy of each patient. Biopsy specimens were inoculated on brain^heart infusion (BHI) agar plates (Oxoid) supplemented with 7% horse blood and antibiotics (vancomycin 6 g ml31 , trimetoprim 2.5 g l31 ). The plates were incubated in microaerobic atmosphere at 37‡C (BBL GasPak System). Colonies of H. pylori were identi¢ed by positive urease, catalase and oxidase tests and characteristic morphology by Gram stain. H. pylori cells were harvested into 500 l TE bu¡er (10 mM Tris and 1 mM EDTA, pH 8.0) and stored at 320‡C until processed. 2.3. Ampli¢cation of cagA To allow PCR based testing for the presence of cagA chromosomal DNA was extracted by the cetyltrimethylammonium bromide (CTAB) method according to the DNA Miniprep protocol of Wilson [8]. The DNA was
dissolved in a ¢nal volume of 50 Wl of TE bu¡er. The primer set used for the detection of the cagA fragment from DNA was F1 (5P-GAT AAC AGG CAA GCT TTT GAG G-3P) and B1 (5P-CTG CAA AAG ATT GTT TGG CAG A-3P). The PCR program for cagA ampli¢cation was: 95‡C, 5 min, 1 cycle; followed by 35 cycles of: 94‡C, 30 s (denaturation), 55‡C, 1 min (annealing), 72‡C, 2 min (polymerization); and 1 cycle at 72‡C for 5 min. A 348-bp internal fragment of cagA was ampli¢ed and the PCR products were resolved on a 1.5% agarose gel. H. pylori NCTC 11637 served as a positive control and sterile distilled water was used as negative control. 2.4. Statistical methods The data were analyzed using SPSS for Windows, version 10.0, and a two-tailed ‘P’ value less than 0.05 was considered signi¢cant. Comparison of measurable data between two groups was done by Student’s t test. Proportions were compared by using chi-square (M2 ) test and Fisher’s exact chi-square test when expected frequencies were less than ¢ve.
3. Results The prevalence of cagA in Turkish pediatric patients was 55.6%. Demographic, clinic and endoscopic ¢ndings of patients are shown in Table 1. There was no statistical signi¢cance in age and gender related to cagAþ and cagA3 patients. Also, symptoms and endoscopic ¢ndings of both groups were not di¡erent from each other (P s 0.05). Duodenal ulcer was observed in one patient who was infected with cagA3 H. pylori strains.
Table 1 The demographic, clinical and endoscopic ¢ndings in cagAþ and cagA3 patients CagA PCR
Gender M/F Age (years) Mean duration of symptoms (months) Symptoms Complaints of abdominal pain Epigastric abdominal pain Periumbilical abdominal pain Unlocalized abdominal pain Vomiting Nausea Halitosis Growth retardation and constipation Hoarse voice Endoscopic ¢ndings Normal Antral nodularity (minimal or prominent) Antral hyperemia Duodenal ulcer Positive urease test
Negative (%)
Positive (%)
Total (%)
9/11 10.9 K 2.6 20.2 K 30.2
14/11 11.2 K 2.5 12.6 K 16.4
45
17 (43.6) 7 5 5 1 ^ ^ 1 1
22 (56.4) 10 9 3 1 1 1 ^ ^
39 (86.7) 17 (37.8) 14 (31.1) 8 (17.8) 2 (4.4) 1 (2.2) 1 (2.2) 1 (2.2) 1 (2.2)
5 (50) 13 (40.6) 1 (50) 1 19 (45.2)
5 (50) 19 (59.4) 1 (50) ^ 23 (54.8)
10 (22.2) 32 (71.1) 2 (4.5) 1 (2.2) 42 (93.3)
FEMSIM 1502 23-4-03
I.N. Salt|k et al. / FEMS Immunology and Medical Microbiology 36 (2003) 147^149
4. Discussion In this study, the prevalence of cagA in Turkish pediatric patients was 55.6%. Although an association between cagAþ H. pylori strains and the presence of severe gastritis and gastric or duodenal ulcer has previously been reported [3^6,9,10], inconsistent results have also been reported [7,11,12]. Kato et al. [7] found that a high prevalence of cagAþ H. pylori strains (80^90%) in children was not associated with nodular gastritis or peptic ulcer disease. Similarly, in our study we did not ¢nd an association between cagA positivity and the severity of symptoms or endoscopic ¢ndings of children. Although presence of the cagA gene in children with duodenal ulcer was high [3,4,6], none of these studies reported a high incidence of peptic ulcer in a¡ected children. In our study only one patient who was infected with cagA3 H. pylori strains had duodenal ulcer and the incidence of peptic ulcer in our patients was 2.2%. These inconsistencies between reports imply that cagA may not be the universal virulence factor. Di¡erences between the results of studies may be due to the genetic or environmental factors that strongly in£uence acquisition and outcome of gastro-duodenal disease caused by H. pylori infection. In conclusion, we were unable to con¢rm an association between cagA status and gastric or duodenal lesions. Further studies to clarify the e¡ect of cagA status in H. pylori infection are needed.
[3]
[4]
[5]
[6]
[7]
[8] [9]
[10]
[11]
References [1] Gold, B.D., Colletti, R.B., Abbott, M., Czinn, S.J., Elitsur, Y., Hassall, E., Macarthur, C., Snyder, J. and Sherman, P.M. (2000) Helicobacter pylori infection in children: recommendations for diagnosis and treatment. J. Pediatr. Gastroenterol. Nutr. 31, 490^497. [2] You, W., Zhang, L., Pan, K., Jiang, J., Chang, Y., Perez-Perez, G.I., Liu, W., Ma, J., Gail, M.H., Blaser, M.J., Fraumeni, J.F. and Xu, G.
[12]
149
(2001) Helicobacter pylori prevalence and cagA status among children in two counties of China with high and low risks of gastric cancer. Ann. Epidemiol. 11, 543^546. Queiroz, D.M.M., Mendes, E.N., Carvalho, A.S.T., Rocha, G.A., Oliveira, A.M.R., Soares, T.F., Santos, A., Cabral, M.M.D.A. and Nogueira, A.M.M.F. (2000) Factors associated with Helicobacter pylori infection by a cagA-positive strain in children. J. Infect. Dis. 181, 626^630. Luzza, F., Pensabene, L., Imeneo, M., Mancuso, M., Giancotti, L., La Vecchia, A.M., Costa, M.C., Strisciuglio, P. and Pallone, F. (2002) Antral nodularity and positive cagA serology are distinct and relevant markers of severe gastric in£ammation in children with Helicobacter pylori infection. Helicobacter 7, 46^52. Lerro, P., Perrucci, V., Morra, I., Lombardo, L., Gaido, F. and Ansaldi, N. (2000) CagA seropositivity and the severity of Helicobacter pylori infection in dyspeptic children. Acta Paediatr. 89, 1312^ 1315. Yahav, J., Fradkin, A., Weisseberg, B., Diver-Haver, A., Shmuely, H. and Jonas, A. (2000) Relevance of CagA positivity to clinical course of Helicobacter pylori infection in children. J. Clin. Microbiol. 38, 3534^3537. Kato, S., Sugiyama, T., Kudo, M., Ohnuma, K., Ozawa, K., Iinuma, K., Asaka, M. and Blaser, M.J. (2000) CagA antibodies in Japanese children with nodular gastritis or peptic ulcer disease. J. Clin. Microbiol. 38, 68^70. Wilson K. (1987) Current Protocols in Molecular Biology. Wiley, New York. Tummuru, M.K., Cover, T.L. and Blaser, M.J. (1993) Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production. Infect. Immun. 61, 1799^1809. Alarcon, T., Martinez, M.J., Urruzuno, P., Cilleruelo, M.L., Madruga, D., Sebastian, M., Domingo, D., Sanz, J.C. and Lopez-Brea, M. (2000) Prevalence of cagA and vacA antibodies in children with Helicobacter pylori associated peptic ulcer compared to prevalence in pediatric patients with active or nonactive chronic gastritis. Clin. Diagn. Lab. Immunol. 7, 842^844. Sheu, S.M., Sheu, S.B., Yang, H.B., Li, C., Chu, T.C. and Wu, J.J. (2002) Presence of iceA1 but not cagA, cagC, cagE, cagF, cagN, cagT, or orf13 genes of Helicobacter pylori is associated with more severe gastric in£ammation in Taiwanese. J. Formos. Med. Assoc. 101, 18^23. Catalano, M., Matteo, M., Barbolla, R.E., Jimenez Vega, D.E., Crespo, O., Leanza, A.G., Toppor, J. and Antelo, P. (2001) Helicobacter pylori vacA genotypes, cagA status and urea-B polymorphism in isolates recovered from an Argentine population. Diagn. Microbiol. Infect. Dis. 41, 205^210.
FEMSIM 1502 23-4-03
www.fems-microbiology.org
The cagA status of Helicobacter pylori isolates from dyspeptic children in Turkey Inci Nur Salt|k a b
a;
º zge Darka Ertunc# b , , Hu«lya Demir a , Doruk Engin b , O Yakut Akyo«n b , Nurten Koc#ak a
Department of Pediatrics, Section of Gastroenterology, Hacettepe University, Faculty of Medicine, 06100 Ankara, Turkey Department of Clinical Microbiology and Microbiology, Hacettepe University, Faculty of Medicine, 06100 Ankara, Turkey Received 4 July 2002; received in revised form 16 October 2002; accepted 4 November 2002 First published online 6 February 2003
Abstract There are inconsistent reports regarding cytotoxin-associated gene A (cagA) status of Helicobacter pylori isolates and the severity of the mucosal lesions in children. The aim of this study was to determine the prevalence of cagAþ strains and to evaluate its correlation with clinic and endoscopic findings. We examined 45 H. pylori strains that were grown on brain^heart infusion agar supplemented with 7% horse blood. Following 72 h of incubation colonies were harvested and bacterial DNA was extracted. Polymerase chain reaction primers F1 and B1 were used to amplify a 348-bp internal fragment of cagA. The prevalence of cagA in Turkish pediatric patients was 55.6%. No association was found between cagA status and the severity of gastro-duodenal lesions. @ 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Helicobacter pylori; cagA; Antral nodularity; Children
1. Introduction Helicobacter pylori infection is usually acquired during childhood and in most children its presence does not lead to clinically apparent disease, even when the organism colonizing the gastric mucosa causes chronic active gastritis. This organism is associated with a signi¢cant proportion of duodenal ulcers and there are epidemiologic data linking chronic H. pylori infection with the development of gastric adenocarcinoma and gastric lymphoma [1,2]. It remains unclear why a minority of H. pylori positive patients develop the disease. It has been suggested that H. pylori strains containing the cytotoxin-associated gene A (cagA) pathogenicity island are more virulent and are associated with the complication of the infection, such as peptic ulcers. The cagA contains a gene that encodes a highly immunogenic outer-membrane protein with a molecular mass of 120^140 kDa and seems to induce an increased in£ammation in the gastric mucosa through release of in-
* Corresponding author. Tel. : +90 (312) 305 1993 and 1994; Fax : +90 (312) 311 7715. E-mail address : [email protected] (I.N. Salt|k).
terleukin-8 (IL-8) by epithelial cells [3]. Results of studies performed in children showed that cagA-positive (cagAþ ) strains of H. pylori caused more severe gastric in£ammation and lower susceptibility to eradication treatment [4^6]. However, in Japanese children, although the prevalence of cagAþ H. pylori strains was high, there was no association with nodular gastritis or peptic ulcer disease [7]. Thus, inconsistent reports exist regarding cagA status of H. pylori isolates and the severity of the mucosal lesions in children. The aim of this study was to determine the prevalence of cagAþ strains in H. pylori isolates from symptomatic children and to evaluate its correlation with clinic and endoscopic ¢ndings.
2. Materials and methods 2.1. Patients H. pylori strains were isolated from 45 patients (22 male ; 48.9%) aged between 6^16 years (mean K S.D.: 11.1 K 2.5 years). Thirty-nine (86.7%) patients had complaints of abdominal pain as the main problem. Other
0928-8244 / 03 / $22.00 @ 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0928-8244(03)00024-5
FEMSIM 1502 23-4-03
148
I.N. Salt|k et al. / FEMS Immunology and Medical Microbiology 36 (2003) 147^149
complaints were halitosis, growth retardation, vomiting and nausea. The mean duration of the symptoms was 15.8 ( K 23.2) months. 2.2. Isolation of H. pylori After informed consent was obtained, an esophagogastroduodenoscopy (EGD) was performed with an Olympus GIF P100 endoscope. Intravenous sedation with midazolam and pethidine was administered to all children before endoscopic examination. Endoscopic ¢ndings were noted for the presence or absence of abnormalities such as antral nodularity, ulcers or erythema. At least four antral gastric biopsies were obtained from each patient for histology, culture and rapid urease test medium (Dio-Helico, Diomed). H. pylori was recovered from one antral biopsy of each patient. Biopsy specimens were inoculated on brain^heart infusion (BHI) agar plates (Oxoid) supplemented with 7% horse blood and antibiotics (vancomycin 6 g ml31 , trimetoprim 2.5 g l31 ). The plates were incubated in microaerobic atmosphere at 37‡C (BBL GasPak System). Colonies of H. pylori were identi¢ed by positive urease, catalase and oxidase tests and characteristic morphology by Gram stain. H. pylori cells were harvested into 500 l TE bu¡er (10 mM Tris and 1 mM EDTA, pH 8.0) and stored at 320‡C until processed. 2.3. Ampli¢cation of cagA To allow PCR based testing for the presence of cagA chromosomal DNA was extracted by the cetyltrimethylammonium bromide (CTAB) method according to the DNA Miniprep protocol of Wilson [8]. The DNA was
dissolved in a ¢nal volume of 50 Wl of TE bu¡er. The primer set used for the detection of the cagA fragment from DNA was F1 (5P-GAT AAC AGG CAA GCT TTT GAG G-3P) and B1 (5P-CTG CAA AAG ATT GTT TGG CAG A-3P). The PCR program for cagA ampli¢cation was: 95‡C, 5 min, 1 cycle; followed by 35 cycles of: 94‡C, 30 s (denaturation), 55‡C, 1 min (annealing), 72‡C, 2 min (polymerization); and 1 cycle at 72‡C for 5 min. A 348-bp internal fragment of cagA was ampli¢ed and the PCR products were resolved on a 1.5% agarose gel. H. pylori NCTC 11637 served as a positive control and sterile distilled water was used as negative control. 2.4. Statistical methods The data were analyzed using SPSS for Windows, version 10.0, and a two-tailed ‘P’ value less than 0.05 was considered signi¢cant. Comparison of measurable data between two groups was done by Student’s t test. Proportions were compared by using chi-square (M2 ) test and Fisher’s exact chi-square test when expected frequencies were less than ¢ve.
3. Results The prevalence of cagA in Turkish pediatric patients was 55.6%. Demographic, clinic and endoscopic ¢ndings of patients are shown in Table 1. There was no statistical signi¢cance in age and gender related to cagAþ and cagA3 patients. Also, symptoms and endoscopic ¢ndings of both groups were not di¡erent from each other (P s 0.05). Duodenal ulcer was observed in one patient who was infected with cagA3 H. pylori strains.
Table 1 The demographic, clinical and endoscopic ¢ndings in cagAþ and cagA3 patients CagA PCR
Gender M/F Age (years) Mean duration of symptoms (months) Symptoms Complaints of abdominal pain Epigastric abdominal pain Periumbilical abdominal pain Unlocalized abdominal pain Vomiting Nausea Halitosis Growth retardation and constipation Hoarse voice Endoscopic ¢ndings Normal Antral nodularity (minimal or prominent) Antral hyperemia Duodenal ulcer Positive urease test
Negative (%)
Positive (%)
Total (%)
9/11 10.9 K 2.6 20.2 K 30.2
14/11 11.2 K 2.5 12.6 K 16.4
45
17 (43.6) 7 5 5 1 ^ ^ 1 1
22 (56.4) 10 9 3 1 1 1 ^ ^
39 (86.7) 17 (37.8) 14 (31.1) 8 (17.8) 2 (4.4) 1 (2.2) 1 (2.2) 1 (2.2) 1 (2.2)
5 (50) 13 (40.6) 1 (50) 1 19 (45.2)
5 (50) 19 (59.4) 1 (50) ^ 23 (54.8)
10 (22.2) 32 (71.1) 2 (4.5) 1 (2.2) 42 (93.3)
FEMSIM 1502 23-4-03
I.N. Salt|k et al. / FEMS Immunology and Medical Microbiology 36 (2003) 147^149
4. Discussion In this study, the prevalence of cagA in Turkish pediatric patients was 55.6%. Although an association between cagAþ H. pylori strains and the presence of severe gastritis and gastric or duodenal ulcer has previously been reported [3^6,9,10], inconsistent results have also been reported [7,11,12]. Kato et al. [7] found that a high prevalence of cagAþ H. pylori strains (80^90%) in children was not associated with nodular gastritis or peptic ulcer disease. Similarly, in our study we did not ¢nd an association between cagA positivity and the severity of symptoms or endoscopic ¢ndings of children. Although presence of the cagA gene in children with duodenal ulcer was high [3,4,6], none of these studies reported a high incidence of peptic ulcer in a¡ected children. In our study only one patient who was infected with cagA3 H. pylori strains had duodenal ulcer and the incidence of peptic ulcer in our patients was 2.2%. These inconsistencies between reports imply that cagA may not be the universal virulence factor. Di¡erences between the results of studies may be due to the genetic or environmental factors that strongly in£uence acquisition and outcome of gastro-duodenal disease caused by H. pylori infection. In conclusion, we were unable to con¢rm an association between cagA status and gastric or duodenal lesions. Further studies to clarify the e¡ect of cagA status in H. pylori infection are needed.
[3]
[4]
[5]
[6]
[7]
[8] [9]
[10]
[11]
References [1] Gold, B.D., Colletti, R.B., Abbott, M., Czinn, S.J., Elitsur, Y., Hassall, E., Macarthur, C., Snyder, J. and Sherman, P.M. (2000) Helicobacter pylori infection in children: recommendations for diagnosis and treatment. J. Pediatr. Gastroenterol. Nutr. 31, 490^497. [2] You, W., Zhang, L., Pan, K., Jiang, J., Chang, Y., Perez-Perez, G.I., Liu, W., Ma, J., Gail, M.H., Blaser, M.J., Fraumeni, J.F. and Xu, G.
[12]
149
(2001) Helicobacter pylori prevalence and cagA status among children in two counties of China with high and low risks of gastric cancer. Ann. Epidemiol. 11, 543^546. Queiroz, D.M.M., Mendes, E.N., Carvalho, A.S.T., Rocha, G.A., Oliveira, A.M.R., Soares, T.F., Santos, A., Cabral, M.M.D.A. and Nogueira, A.M.M.F. (2000) Factors associated with Helicobacter pylori infection by a cagA-positive strain in children. J. Infect. Dis. 181, 626^630. Luzza, F., Pensabene, L., Imeneo, M., Mancuso, M., Giancotti, L., La Vecchia, A.M., Costa, M.C., Strisciuglio, P. and Pallone, F. (2002) Antral nodularity and positive cagA serology are distinct and relevant markers of severe gastric in£ammation in children with Helicobacter pylori infection. Helicobacter 7, 46^52. Lerro, P., Perrucci, V., Morra, I., Lombardo, L., Gaido, F. and Ansaldi, N. (2000) CagA seropositivity and the severity of Helicobacter pylori infection in dyspeptic children. Acta Paediatr. 89, 1312^ 1315. Yahav, J., Fradkin, A., Weisseberg, B., Diver-Haver, A., Shmuely, H. and Jonas, A. (2000) Relevance of CagA positivity to clinical course of Helicobacter pylori infection in children. J. Clin. Microbiol. 38, 3534^3537. Kato, S., Sugiyama, T., Kudo, M., Ohnuma, K., Ozawa, K., Iinuma, K., Asaka, M. and Blaser, M.J. (2000) CagA antibodies in Japanese children with nodular gastritis or peptic ulcer disease. J. Clin. Microbiol. 38, 68^70. Wilson K. (1987) Current Protocols in Molecular Biology. Wiley, New York. Tummuru, M.K., Cover, T.L. and Blaser, M.J. (1993) Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production. Infect. Immun. 61, 1799^1809. Alarcon, T., Martinez, M.J., Urruzuno, P., Cilleruelo, M.L., Madruga, D., Sebastian, M., Domingo, D., Sanz, J.C. and Lopez-Brea, M. (2000) Prevalence of cagA and vacA antibodies in children with Helicobacter pylori associated peptic ulcer compared to prevalence in pediatric patients with active or nonactive chronic gastritis. Clin. Diagn. Lab. Immunol. 7, 842^844. Sheu, S.M., Sheu, S.B., Yang, H.B., Li, C., Chu, T.C. and Wu, J.J. (2002) Presence of iceA1 but not cagA, cagC, cagE, cagF, cagN, cagT, or orf13 genes of Helicobacter pylori is associated with more severe gastric in£ammation in Taiwanese. J. Formos. Med. Assoc. 101, 18^23. Catalano, M., Matteo, M., Barbolla, R.E., Jimenez Vega, D.E., Crespo, O., Leanza, A.G., Toppor, J. and Antelo, P. (2001) Helicobacter pylori vacA genotypes, cagA status and urea-B polymorphism in isolates recovered from an Argentine population. Diagn. Microbiol. Infect. Dis. 41, 205^210.
FEMSIM 1502 23-4-03