| Abstract|| |
Background/Aim: It is important to eradicate Helicobacter pylori at an early stage in patients during childhood to potentially prevent the development of H. pylori-related diseases. Studies have demonstrated that the virulence genotype of H. pylori influences the efficacy of eradication therapy. The efficacy of triple therapy has decreased significantly, which has seriously affected the clinical outcome of children with H. pylori infection. In this study we aimed to investigate the influence of virulence genotypes of H. pylori on triple eradication therapy in children.
Patients and Methods: H. pylori strains were isolated from the gastric antrum mucosa in children with upper gastrointestinal symptoms. Polymerase chain reaction (PCR) was conducted to determine the H. pylori cagA, vacA, and iceA genotypes. All patients with H. pylori infection were administered 14-day triple therapy. After drug withdrawal for at least 4 weeks, the 13C-urea breath test (13C-UBT) was used to observe the therapeutic effect of H. pylori eradication. The eradication rates were evaluated by intention-to-treat (ITT) and per-protocol (PP) analyses.
Results: A total of 107 patients were enrolled in this study. Nine patients were lost to follow-up, and 98 patients were administered eradication therapy. Based on ITT and PP analyses, the H. pylori eradication rate was 64.5% (69/107) and 70.4% (69/98), respectively. Among the successful eradication groups, the cagA-positive, vacA s1a, vacA s1c, vacA m1, vacA m2, iceA 1, and iceA 2 genes were identified in 72.8%, 68.1%, 76.9%, 60.0%, 74.6%, 71.8%, and 75.0% of strains, respectively. Of the unsuccessful eradication groups, the cagA-positive, vacA s1a, vacA s1c, vacA m1, vacA m2, iceA 1, and iceA 2 genes were identified in 27.2%, 31.9%, 23.1%, 40.0%, 25.4%, 28.2%, and 25.0% of strains, respectively. No statistically significant differences were noted in the detection rate of the H. pylori genotypes between the H. pylori successful and unsuccessful eradication groups (P > 0.05).
Conclusions: The cagA, vacA, and iceA genotypes of H. pylori are not associated with the efficacy of omeprazole-based triple therapy on the eradication of H. pylori infection in children.
Keywords: Child, Helicobacter pylori, polymerase chain reaction, triple therapy, virulence genotype
|How to cite this article:|
Zhang SH, Zhu X, Li BM, Li H. The effect of virulence genotypes of Helicobacter pylori on eradication therapy in children. Saudi J Gastroenterol 2018;24:249-54
|How to cite this URL:|
Zhang SH, Zhu X, Li BM, Li H. The effect of virulence genotypes of Helicobacter pylori on eradication therapy in children. Saudi J Gastroenterol [serial online] 2018 [cited 2018 Nov 18];24:249-54. Available from: http://www.saudijgastro.com/text.asp?2018/24/4/249/229991
| Introduction|| |
Helicobacter pylori (H. pylori) is associated with various diseases of the upper gastrointestinal tract, such as gastritis, peptic ulcers, and mucosa-associated lymphoid tissue in children. It is important to eradicate H. pylori at an early stage in patients during childhood to potentially prevent the development of H. pylori-related diseases. The main virulence genes of H. pylori include cagA, vacA, and iceA. At present, the recommended therapy for eradicating H. pylori in pediatric patients is triple therapy, which consists of a proton pump inhibitor (PPI) and two other antibiotics. The efficacy of triple therapy has decreased in children. The reasons for failure in eradication mainly include H. pylori strain factors, host factors, environmental factors, inappropriate treatment, and low compliance to therapy. This study aimed to investigate the influence of H. pylori virulence genotypes on 14-day omeprazole-based triple therapy with two antibiotics by detecting the cagA, vacA, and iceA status of H. pylori in children.
| Patients and Methods|| |
From July 2014 to September 2015, 107 patients who suffered from abdominal pain, vomiting, belching, and gastrointestinal bleeding and who underwent gastroscopy were enrolled in this study. After informed consent was obtained, gastric biopsies were collected by a clinical gastroenterologist. The exclusion criteria were as follows: (1) patients who had taken any PPI, H2 receptor antagonists, bismuth salts, or antibiotics for at least 4 weeks prior to the time of their enrolment in the study; (2) patients whose condition was complicated by severe heart, lung, blood, liver, or kidney dysfunction; and (3) patients who would not comply to the study. The ethics committee of the Children's Hospital of Jiangxi approved the study, NO. JXSETYY-2016003. Informed consent was obtained from the parents or guardians of the children enrolled in the study.
H. pylori isolation and culture
Gastric mucosal biopsy specimens were used for H. pylori culture. The biopsies from each patient were cultured on the surface of Karmali agar (Oxoid, Basingstoke, Hampshire, England) plates. The cultured plates were incubated in microaerobic atmosphere conditions (37°C, 5% oxygen, 10% carbon dioxide, and 85% nitrogen) for 3 to 7 days. After culture, smooth, neat, gray, circular, translucent colonies approximately 0.5 to 2 mm in diameter were identified as H. pylori based on a rapid urease test and modified Giemsa staining; consistent positive results between the two tests was required. The bacterial isolates were preserved in brain heart infusion broth enriched with 20% glycerol and 10% inactivated horse serum and were stored at −80°C. ThestandardNCTC11639 H. pylori strain was donated by the Institute of Digestive Diseases, the First Affiliated Hospital of Nanchang University.
H. pylori eradication therapy
Patients received 14-day triple therapy consisting of omeprazole (0.6–0.8 mg/kg, bid), amoxicillin (50 mg/kg/day, bid), clarithromycin (15–20 mg/kg/day, bid), or metronidazole (20 mg/kg/day, bid). For patients who were allergic to penicillin, amoxicillin was replaced with metronidazole. For patients with gastrointestinal bleeding and peptic ulcers, omeprazole (0.6–0.8 mg/kg, qd) was administered for an additional 2–4 weeks after the 14-day treatment.
PCR amplification of H. pylori virulence genotypes
Genomic DNA was extracted from H. pylori strains isolated from gastric biopsy specimens using the QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was stored at −20°C until it was used for polymerase chain reaction (PCR) amplification. PCR was performed on purified genomic DNA from all H. pylori isolates to examine the presence of virulence genotypes, including cagA, vacA, and iceA. Primers were designed based on published papers and are listed in [Table 1].,, The primer sequences for H. pylori DNA were synthesized by GenScript Co. Ltd. (Nanjing, China). Each PCR was performed in a total volume of 25 μl. The final reaction mixture contained 500 ng of genomic DNA, 8.5 μl of distilled water, and 12.5 μl of 2 × Taq PCR master mix (TIANGEN, KT201, Beijing, China), including 0.1 U/μl Taq Polymerase, 500 μmol/L of each dNTP, 20 mmol/L Tris-HCl (pH 8.3), 100 mmol/L KCl, 3 mmol/L MgCl2, and 2 μl of each primer (forward and reverse mixture). The PCR amplification conditions were as follows: pre-denaturation was performed at 95°C for 5 min; 30 cycles of 30 s at 94°C, 30 s at 55°C (for ureA, cagA, vacA s1, vacA s1a, vacA s1b, vacA m1, vacA m2, iceA1, and iceA2) or 59°C (for vacA s1c and vacA s2) for annealing; and a final extension at 72°C for 7 min. The final reactions were stored at 4°C. PCR products were electrophoresed on a 2.0% (w/v) agarose gel (Sigma, USA) that was stained with 0.5 μg/ml ethidium bromide and developed under UV light (Bio-Rad, USA) according to the standard procedures. A 100-bp DNA ladder (TIANGEN, D2000, Beijing, China) served as a molecular size marker.
|Table 1: PCR primers for the amplification of H. pylori cagA, vacA and iceA sequences|
Click here to view
At least 4 weeks after the completion of treatment, bacterial eradication was assessed using a 13C-urea breath test (13C-UBT). 13C-UBT employed a 13C-urea 75-mg capsule (Zhonghe Headway Bio-sci and Tech Co, Ltd, Shenzhen, China). When the 30-min Delta over baseline value was 4 or more, the patient was determined to be positive for H. pylori. A negative result was regarded as an eradication success, whereas a positive result was regarded as an eradication failure.
Statistical analysis was performed using (SPSS Inc., Chicago, IL, USA). The eradication rates were evaluated by intention-to-treat (ITT) and per-protocol (PP) analyses. Chi-square test was used for comparisons between the groups. A P value of <0.05 was considered statistically significant.
| Results|| |
Characteristics of the study group
Of all the 107 children, 98 completed eradication therapy and received 13C-UBT after treatment. Among these patients, 77 were males and 21 were female. The patients ranged from 4 to 14 years old. According to the endoscopy diagnoses, 29 patients had chronic gastritis, 12 had duodenal bulb inflammation, and 57 had peptic ulcers (3 had gastric ulcers and 54 had duodenal ulcers). Nine patients did not complete the treatment. Among these patients, 2 were too young to receive eradication therapy, 1 patient discontinued treatment due to a rash, 4 patients did not receive 13C-UBT after treatment, and 2 patients were lost to follow-up.
Eradication of H. pylori
Among 98 patients, 69 had a negative result for 13C-UBT and were regarded as eradication success. The eradication rates according to ITT and PP analysis were 64.5% (69/107) and 70.4% (69/98), respectively.
Analysis of cagA, vacA, and iceA genotypes
Among 98 strains, the cagA subtypes were detected in 92 strains of H. pylori isolates. The number of strains positive for vacA s1a, vacA s1c, vacA m1, and vacA m2 subtypes were 72, 26, 30, and 67 strains, respectively. One strain was both vacA m1 and vacA m2 positive. The number of strains positive for vacA s1a/m1, vacA s1a/m2, vacA s1c/m2, and vacA s1c/m1 subtypes was 26, 49, 4, and 18 strains, respectively. However, the vacA s1b and vacA s2 subtypes were not detected in any samples. In addition, the number of strains positive for iceA 1 and iceA 2 subtypes was 78 and 8 strains, respectively, whereas 8 strains were both iceA 1 and iceA 2 positive [Table 2].
Association of H. pylori genotypes with eradication rates
Among the successful eradication groups, the cagA, vacA s1a, vacA s1c, vacA m1, vacA m2, iceA 1, and iceA 2 genes were identified in 72.8%, 68.1%, 76.9%, 60.0%, 74.6%, 71.8%, and 75.0% of strains, respectively. Of the unsuccessful eradication groups, the cagA-positive, vacA s1a, vacA s1c, vacA m1, vacA m2, iceA 1, and iceA 2 genes were identified in 27.2%, 31.9%, 23.1%, 40.0%, 25.4%, 28.2%, and 25.0% of strains, respectively. No statistically significant differences in the prevalence of H. pylori genotypes were noted between the successful and unsuccessful eradication groups (P > 0.05) [Table 3].
|Table 3: Relationship between the detection rate of H. pylori genotypes and treatment outcome|
Click here to view
| Discussion|| |
H. pylori is a Gram-negative, spiral, microaerophilic bacterium. The bacteria can adhere to the surface of the gastric mucosal epithelium and the bottom layer of the gastric mucosa, and the infection status can be lifelong if the infected person does not undergo standardized treatment. At present, the recommended therapy to eradicate H. pylori in children involves triple therapy that consists of a PPI and two additional antibiotics. In regard to eradication therapy, PPIs have an anti-H. pylori effect. The bactericidal effect of antibiotics depends on strong acid inhibition of PPI, whereas PPIs and antibiotics have synergistic bactericidal effects. Thus, PPI-based triple therapy may increase the efficacy of H. pylori eradication therapies. Given the development of this triple therapy and continued widespread use of antibiotics in general practice in recent years, the failure rate of this triple therapy has increased among children. H. pylori eradication treatment may fail for a number of reasons, including antibiotic resistance, bacterial virulence factors, environmental factors, different diseases, and low compliance to therapy.,,,,H. pylori virulence genes include genes such as vacA, cagA, and iceA. The cytotoxin-associated gene A (cagA) gene, which encodes cytotoxin-associated A protein (CagA), enters host epithelial cells of the stomach using a type IV secretion system, undergoes phosphorylation, interferes with host cell signalling pathways, and produces severe inflammation and tissue damage. Vacuolating cytotoxin gene A (vacA) encodes vacuolating cytotoxin A (VacA). The vacA genetic structure has alleles of the mosaic structure, including the signal (s) and middle (m) regions. The “s” and “m” regions are divided into s1 (s1a, s1b, and s1c) or s2 and m1 or m2 subtypes, respectively. The induced by contact with epithelium (iceA) gene has two main allelic variants – iceA1 and iceA2. Several studies have reported that the virulence genotype of H. pylori may play an important role in the development of gastrointestinal disease.,
This study focused on 107 children who were positive for H. pylori culture and were cured with a 14-day triple therapy with omeprazole, amoxicillin, clarithromycin, or metronidazole. In total, 98 patients completed the treatment, and 69 exhibited successful eradication based on 13C-UBT after treatment. The eradication rate according to ITT and PP analyses were 64.5% (69/107) and 70.4% (69/98), respectively. Over the past decades, standard triple therapy contributed to a successful eradication rate of greater than 90% in children. However, successful eradication rates have significantly declined. The emergence of H. pylori-resistant strains is one of the most important causes of the reduction in the eradication rate of triple regimens with a PPI plus two antibiotics. At present, the most common resistance to antibiotics worldwide among affected children involve metronidazole and clarithromycin, whereas the resistance rate of amoxicillin is lower., In China, the rates of resistance to metronidazole and clarithromycin were increased among children infected with H. pylori with resistance rates of 49.2% and 34.9%, respectively. The amoxicillin resistance rate was 6.2%. In Japan, the rates of resistance to metronidazole and clarithromycin were 43.3% and 21.9%, respectively, among children infected with H. pylori. In Turkey, the rates of resistance to metronidazole and clarithromycin among children infected with H. pylori were 48.4% and 30.1%, respectively; resistance to amoxicillin was not observed.
The cagA, vacA, and iceA genes are the major virulence genes of H. pylori. Studies have assessed the relationship between H. pylori genes and eradication therapy. The results of this study demonstrated that, among the groups that underwent successful eradication, the cagA, vacA s1a, vacA s1c, vacA m1, vacA m2, iceA 1, and iceA 2 genes were identified in 72.8%, 68.1%, 76.9%, 60.0%, 74.6%, 71.8%, and 75.0% of strains, respectively. Of the groups that did not undergo successful eradication, the cagA, vacA s1a, vacAs1c, vacA m1, vacA m2, iceA 1, and iceA 2 genes were identified in 27.2%, 31.9%, 23.1%, 40.0%, 25.4%, 28.2%, and 25.0% of strains, respectively. No statistically significant differences in the detection rate of H. pylori genotypes were noted between the successful and unsuccessful H. pylori eradication groups (P > 0.05). These results suggest that the cagA, vacA, and iceA genotypes of H. pylori may have no relation to the efficacy of omeprazole-based 14-day triple therapy in children. A study published by Van Doorn et al. suggested that the eradication rates of H. pylori were increased for cagA-positive and vacA s1 H pylori strains, a finding that is consistent with the increased eradication rate observed among ulcer patients compared with functional dyspepsia patients. Khan et al. demonstrated that the alarming levels of antibiotic resistance were associated with the cagA gene in H. pylori strains. The results of this study are inconsistent with previous reports, which may be related to the differences in H. pylori infection rates between children and adults, and the different regions and H. pylori strain polymorphisms in different regions. The cagA-negative and vacA s2 H. pylori strain may increase the risk of H. pylori eradication failure, suggesting that cagA and vacA genotypes play an important role in H. pylori eradication therapy. A possible reason is that cagA-positive strains can induce severe inflammatory reactions in the gastric mucosa. When gastric mucosal inflammation is obvious, the blood flow in the mucosa is rich, which is beneficial to the efficacy of antibiotics in the gastric mucosa compared with the cagA-negative strains. In the gastric mucosa, IL-1β can inhibit gastric acid secretion. The cagA-positive strain noted in children produces significantly increased IL-1β in the gastric mucosa compared with cagA-negative strains, which produce less gastric acid secretion. This feature makes the area more accessible to antibiotics and conducive to curing H. pylori. The vacA s1 and vacA m1 genotypes of H. pylori have been associated with severe gastric inflammation, produce a large amount of toxins, and induce increased vacuolating activity in gastric epithelial cells. Thus, this genotype enhances the antibacterial effect of antibiotics, whereas the vacA s2 and vacA m2 genotypes produce minimal or no toxins.,
In conclusion, in areas of low (<20%) clarithromycin resistance, triple therapy involving PPI and two antibiotics should be recommended as first-line treatment for children with H. pylori infection. The results of this study indicate that the eradication rate of H. pylori in children has declined considerably compared with previous studies. In addition, the cagA, vacA, and iceA genotypes of H. pylori are not associated with the efficacy of 14-day omeprazole-based triple therapy on the eradication of H. pylori infection in children. Since the sample size in this study is limited, a multicenter, randomized controlled trial with a large sample size is needed to further address the issue.
We would like to thank Yan Liu, You-Ju Li, and Lan Wei for their help with collecting tissue specimens from the gastric antrum in children used in this study. The research is supported by a grant from the Jiangxi Provincial Department of Science and Technology.
| References|| |
Oderda G, Marietti M, Pellicano R. Diagnosis and treatment of Helicobacter pylori
infection in pediatrics: Recommendation for 2014 clinical practice. Minerva Pediatr 2015;67:517-24.
Malfertheiner P, Megraud F, O'Morain CA, Gisbert JP, Kuipers EJ, Axon AT, et al
. Management of Helicobacter pylori
infection-the Maastricht V/Florence Consensus Report. Gut 2017;66:6-30.
De Gusmão VR, Nogueira Mendes E, De Magalhães Queiroz DM, Aguiar Rocha G, Camargos Rocha AM, Ramadan Ashour AA, et al
. vacA genotypes in Helicobacter pylori
strains isolated from children with and without duodenal ulcer in Brazil. J Clin Microbiol 2000;38:2853-7.
Yamaoka Y, Kodama T, Gutierrez O, Kim JG, Kashima K, Graham DY. Relationship between Helicobacter pylori
iceA, cagA, and vacA status and clinical outcome: Studies in four different countries. J Clin Microbiol 1999;37:2274-9.
Ito Y, Azuma T, Ito S, Miyaji H, Hirai M, Yamazaki Y, et al
. Analysis and typing of the vacA gene from cagA-positive strains of Helicobacter pylori
isolated in Japan. J Clin Microbiol 1997;35:1710-4.
Sugano K, Tack J, Kuipers EJ, Graham DY, El-Omar EM, et al
. Kyoto global consensus report on Helicobacter pylori
gastritis. Gut 2015;64:1353-67.
Gisbert JP, McNicholl AG. Optimization strategies aimed to increase the efficacy of H. pylori eradication therapies. Helicobacter 2017;22.
Gong EJ, Yun SC, Jung HY, Lim H, Choi KS, Ahn JY, et al
. Meta-analysis of first-line triple therapy for helicobacter pylori
eradication in Korea: Is it time to change? J Korean Med Sci 2014;29:704-13.
Lee JY, Kim N, Kim MS, Choi YJ, Lee JW, Yoon H, et al
. Factors affecting first-line triple therapy of Helicobacter pylori
including CYP2C19 genotype and antibiotic resistance. Dig Dis Sci 2014;59:1235-43.
Murakami TT, Scranton RA, Brown HE, Harris RB, Chen Z, Musuku S, et al
. Management of Helicobacter Pylori
in the United States: Results from a national survey of gastroenterology physicians. Prev Med 2017;100:216-22.
Shiota S, Nguyen LT, Murakami K, Kuroda A, Mizukami K, Okimoto T, et al
. Association of helicobacter pylori
dupA with the failure of primary eradication. J Clin Gastroenterol 2012;46:297-301.
Roma E, Miele E. Helicobacter pylori
Infection in Pediatrics. Helicobacter 2015;20(Suppl 1):47-53.
Ki MR, Hwang M, Kim AY, Lee EM, Lee EJ, Lee MM, et al
. Role of vacuolating cytotoxin VacA and cytotoxin-associated antigen CagA of Helicobacter pylori
in the progression of gastric cancer. Mol Cell Biochem 2014;396:23-32.
Almeida N, Donato MM, Romãozinho JM, Luxo C, Cardoso O, Cipriano MA, et al
. Correlation of Helicobacter pylori
genotypes with gastric histopathology in the central region of a South-European country. Dig Dis Sci 2015;60:74-85.
Gościniak G, Biernat M, Grabińska J, Bińkowska A, Poniewierka E, Iwańczak B. The antimicrobial susceptibility of Helicobacter pylori
strains isolated from children and adults with primary infection in the Lower Silesia Region, Poland. Pol J Microbiol 2014;63:57-61.
Vanderpas J, Bontems P, Miendje Deyi VY, Cadranel S. Follow-up of Helicobacter pylori
infection in children over two decades (1988-2007): Persistence, relapse and acquisition rates. Epidemiol Infect 2014;142:767-75.
Li L, Ke Y, Yu C, Li G, Yang N, Zhang J, et al
. Antibiotic resistance of Helicobacter pylori
in Chinese children: A multicenter retrospective study over 7 years. Helicobacter 2017;22.
Okuda M, Kikuchi S, Mabe K, Osaki T, Kamiya S, Fukuda Y, et al
. Nationwide survey of Helicobacter pylori
treatment for children and adolescents in Japan. Pediatr Int 2017;59:57-61.
Maçin S, Demir H, Özen H, Yüce A, Akyön Y. Determination of Helicobacter pylori
antibiotic resistance patterns in pediatric gastroenterology patients: The Hacettepe experience. Turk J Pediatr 2015;57:254-7.
Van Doorn LJ, Schneeberger PM, Nouhan N, Plaisier AP, Quint WG, De Boer WA. Importance of Helicobacter pylori
cagA and vacA status for the efficacy of antibiotic treatment. Gut 2000;46:321-6.
Khan A, Farooqui A, Manzoor H, Akhtar SS, Quraishy MS, Kazmi SU. Antibiotic resistance and cagA gene correlation: Looming crisis of Helicobacter pylori
. World J Gastroenterol 2012;18:2245-52.
Sugimoto M, Yamaoka Y. Virulence factor genotypes of Helicobacter pylori
affect cure rates of eradication therapy. Arch Immunol Ther Exp (Warsz) 2009;57:45-56.
Russo F, Berloco P, Cuomo R, Caruso ML, Di Matteo G, Giorgio P, et al
. Helicobacter pylori
strains and histologically-related lesions affect the outcome of triple eradication therapy: A study from southern Italy. Aliment Pharmacol Ther 2003;17:421-8.
Dr. Xuan Zhu
Department of Gastroenterology, The First Affiliated Hospital, Nanchang University, 17 Yongwaizheng Street, Nanchang, - 330006, Jiangxi Province
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]