ABSTRACT

Objective: This study aimed to determine the epidemiological characteristics of Helicobacter pylori (H. pylori) infection at the genotype level in patients presenting with dyspeptic complaints, and to show the distribution of virulence factors, importance of intrafamilial transmission, as well as the distribution of resistance to macrolide and quinolone antibiotics.

Methods

The study comprised 110 patients with dyspeptic complaints who were admitted to our hospital between January 13, 2015, and December 31, 2016. Through histopathology, culture, and glmM-polymerase chain reaction (PCR) techniques, we detected H. pylori. The vacA, cagA, and cagE genes were determined in patients with positive PCR results were positive.

Results

H. pylori strains and clinical results were not found to be significantly correlated in the study. Both genetic variants A2142G and A2143G were discovered to be present in the individuals. Eight patients had clarithromycin resistance (34.7%). All patients with a positive A2142G mutation and 55% of patients with a positive A2143G mutation were found to have clarithromycin resistance. Levofloxacin resistance was present in only one (4.3%) patient who could produce H. pylori in culture.

Conclusion

Approximately 1/3 of the children with dyspeptic complaints were positive for H. pylori infection. The most common genotype was observed to be vacAs2. Even individuals with at least one of the genetic mutations A2142G and A2143G have the potential for antibiotic resistance. High resistance was found against clarithromycin in the standard triple therapy regimen used in children for treating H. pylori infection.

INTRODUCTION

Helicobacter pylori (H. pylori) is the most important cause of gastroduodenal pathology, ranging from non-ulcer dyspepsia to gastric ulcer and mucosa-associated lymphoid tissue lymphoma, to atrophic gastritis to lower-gastrointestinal system (GIS) carcinomas (1-4). Epidemiological studies have suggested that H. pylori is usually transmitted from the family in childhood; more than one strain initiated the infection in the first transmission, but one of the strains adapted to the gastric mucosa in the subsequent period (3).

Although many factors belonging to bacteria and the host have been suggested in the possible relationship between gastroduodenal pathology and H. pylori, it can be said most crucial evidence in this context is the clinical cure after H. pylori eradication therapy. The frequent occurrence of reactivations due to resistance to various antibiotic regimens applied in recent years in bacteria can be considered the second vital data that strengthens the relationship. Developing more rational treatments will only be possible by identifying colonized strains and knowing the movement of resistance in society by identifying resistant strains.

This study aimed to determine the epidemiological characteristics of H. pylori infection at the genotype level in childhood gastroduodenal pathologies. In addition, this study aimed to evaluate the initial period of the H. pylori-human relationship, the characteristics of the colonized strain(s), the distribution of virulence factors, the importance of intra-familial transmission, and the primary/secondary resistance distribution to macrolide and quinolone group antibiotics.

MATERIALS AND METHODS

A total of 110 cases admitted with dyspeptic complaints between 13 February 2015 and 1 December 2016 were included in the study. The criteria for inclusion in the study; cases between the ages of 3 and 18 years were identified as cases in which upper GIS endoscopy was planned due to dyspepsia and patients who had not previously received eradication treatment for H. pylori. Patients with upper gastrointestinal tract bleeding, patients with a history of gastroduodenal surgery, patients who did not agree to participate in the study, patients with a history of antibiotic and/or proton pump inhibitor use up to 1 month before the study, and patients with chronic diseases were excluded from the study.

After upper GIS endoscopy were performed and the findings were recorded, three biopsy samples were collected from the antrum region of the patients included in the study. The first sample was used for pathological examination, the second was used for H. pylori isolation in culture medium and for determining antibiotic susceptibility, and the third was used for genotypic examination. The E-test was performed to assess antibiotic susceptibility to H. pylori strains isolated in culture, and the minimum inhibitory concentration (MIC) values were determined. In our study, clarithromycin and levofloxacin E-test strips (Biotests, Ankara, Türkiye, and Liofilche, Italy) were used. The MICs were accepted as determined by the European Committee on Antimicrobial Susceptibility Testing in 2016 (5). For genotypic examinations, DNA extraction was performed from gastric biopsy samples, and these extracts were first examined by polymerase chain reaction (PCR) with the help of specific primers to determine the glmM gene sequence. The samples with positive glmM gene were accepted as H. pylori-positive, and their DNA extracts were evaluated by PCR for the detection of vacA, cagA, and cagE, PCR-RFLP for the detection of clarithromycin resistance, and DNA Sequence analysis for the detection of levofloxacin resistance. Additionally, to detect familial genetic and pathogenic inheritance, upper GIS endoscopy was performed by an adult gastroenterologist in 7 parents of 6 H. pylori-positive cases.

Ethics Committee approval was obtained from the Non-Interventional Clinical Research Ethics Committee of Çukurova University Faculty of Medicine (approval number: 37, date: 05.12.2014). The relatives of all the children included in the study were interviewed. After providing information about the interventional procedures to be applied to patients, the purpose of the research, and where to use the data, verbal and written consent was obtained.

Statistical Analysis

Statistical evaluations were performed using the SPSS 21.0 package program. Descriptive data are presented as percentage distributions. The chi-square test was used to compare the gender distribution, endoscopic and genotypic findings. Fischer’s exact test was used to compare categorical variables. The statistical significance level was established as 0.05 in all tests.

RESULTS

The mean age of the cases was 12.2±4.39 years (3-18 years), as 38 were male (34.5%) and 72 were female (65.5%). H. pylori was found to be positive in 30 cases (27.3%) by PCR method. 18.8% of H. pylori-positive patients were between 3-6 years old, 33.3% were between 7-12 years old, and 25.9% were between 13-18 years old. No statistically significant difference was found between the H. pylori-positive and H. pylori-negative groups in terms of gender and age (p=0.235, p=0.519, respectively).

Of 30 PCR-positive patients, 29 (96.6%) were found to be vacA positive, and vacAs1+cagA+cagE association was found in 16.6% (Table 1).

Among the virulence factors, cagA was found to be positive in 50% of vacAs1-positive patients and 29.4% of vacAs2-positive patients. When the association between vacA subtypes and cagA was examined, no statistically significant difference was found (p=0.390). It was found that 58.3% of the vacAs1-positive patients were cagE-positive. No association was observed between vacAs2 and cagE in any of the patients. When the distribution of virulence genes in H. pylori PCR-positive cases was examined, no statistically significant relationship was found (p>0.05).

It was observed that vacAs2, one of the less virulent vacA alleles, was found in all cases with normal histopathology, cagA was positive in 66.6% of these cases, and vacAs1 and cagE were not found in these cases. In patients with active chronic gastritis, the vacAs1, vacAs2, cagA, and cagE virulence genes were found to be 44.4%, 51.8%, 33.3%, and 25.9%, respectively.

Antibiogram was performed in 23 (20.9%) cases with positive H. pylori culture. Clarithromycin resistance was found in 8 (34.7%) patients, and no significant difference was found between genders in terms of clarithromycin resistance (p=0.342). Of clarithromycin-resistant cases, vacAs1 was found to be positive in 50%, vacAs2 in 36.4%, cagA in 40%, and cagE in 66.7%. However, no statistically significant intergroup difference was observed regarding the relationship between virulence genes and clarithromycin resistance (Table 2). Levofloxacin resistance was observed in only one case (4.3%), and vacAs1 was positive; vacAs2, cagA, and cagE were negative in this case.

When the A2142G gene mutation distribution was examined according to the age groups of the patients, it was found that there were no genetic mutations between the ages of 3-6 and the presence of genetic mutations at a rate of 16.7% between the ages of 7-12 and 20% between the ages of 13-18. When the A2143G gene mutation distribution was examined by age groups, it was observed with a rate of 33.3% between ages 3-6, 25% between ages 7-12, and 53.3% between ages 13-18. Per age group, no statistically significant difference was found in terms of A2142G and A2143G gene mutation positivity (p=0.698, p=0.318, respectively).

It was found that all patients with the positive A2142G mutation were clarithromycin-resistant, and clarithromycin resistance was found to be significantly higher than those who were negative (p=0.023). No significant difference was found in clarithromycin resistance between the A2143G mutation-positive patients than the negative ones (p=0.054) (Table 3). Both A2142G and A2143G genetic mutations were found to be positive in a single patient with levofloxacin resistance.

In our study, endoscopy was performed in 7 parents of 6 PCR-positive patients with clinical symptoms, and different H. pylori strains were found in the children and parents (Table 4).

DISCUSSION

H. pylori is one of the most common pathogens in humans (6). In addition to virulence factors such as urease activity and motility, immunogenic cross-reactivity in gastric cells plays an essential role in the pathogenesis of H. pylori (7). In addition, it is thought that some of the H. pylori virulence genes increase the risk of developing gastric lesions, and the pathogenic effect of bacteria virulence factors is affected by many factors belonging to the environment and host (8).

In our country, the number of studies evaluating H. pylori virulence genes in children is extremely low. Looking at vacA isolates from virulence genes, in 2013, Ozbey et al. (9) reported that 91.8% of vacAs1 isolates in H. pylori-positive children, and in 2014, Karabiber et al. (10) reported that 81.6% of them were vacAs1 subtype, and the most common vacA subtype was vacAs1a/m2, at a rate of 32.7%. vacAs1/m1 was the most common allele in 85% of symptomatic children in Venezuela and vacAs1 in 82.5% of asymptomatic children in Brazil (11, 12). In a study conducted by Erdoğdu et al. (13) in 120 children, it was found that 70.1% of H. pylori isolates had vacAs1a, 2.8% had vacAs1b, and 27.1% had the vacAs2 subtype, and reported that vacA genotype was not associated with endoscopic findings. While the most common genotype in Iranian children is vacAs1/m2; it was found that nodular gastritis is common in endoscopic findings, and this is significantly associated with the presence of vacAm1 (14).In our study, the vacAs1 allele was found to be positive in 40% of H. pylori-positive cases, and the vacAs2 allele, which is known to be less virulent, in 56.6%. Compared with these two studies, it was observed that the vacAs2 allele was higher in our study. In our research, no statistically significant difference was found in the evaluation of the vacAs1 and vacAs2 genotypes according to endoscopic findings and histopathological data.

In our study, cagA was found with a rate of 36.7%. In symptomatic children, H. pylori cagA strains and serum cagA antibodies are found at the rate of 33% -80% (15-17). In a study by Kato et al. (18) conducted on 25 children with the ulcer, it was reported that cagA was detected in 81.8% of asymptomatic children, 93.3% of patients with gastritis, and 80% of patients with gastric ulcers.In a study conducted by Saltik et al. (19), 45 children were evaluated for abdominal pain and tested only for cagA, which is one of the virulence factors. They found cagA positivity at a rate of 55.6%, and they reported that there was no relationship between the severity of gastrointestinal findings.

When cagE, a virulence gene, was examined, 23.3% of the 30 PCR-positive patients in our study were found to be cagE positive. A vacAs1+cagE association was found in 6.6% of these, and a vacAs1+cagA+cagE association was found in 16.6%. Because there were not enough studies on this subject in children, cagE positivity was found at a low rate in our study compared to adult studies conducted in our country, whereas a similar rate of cagE positivity was found when compared to the study conducted by Ozbey et al. (9).

In our study, active chronic gastritis was detected in 90% of H. pylori PCR-positive 30 patients, and histopathological evaluation of 14.3% of these patients was found to be normal. Among these H. pylori PCR-positive cases, it was observed that all of the vacA alleles with normal histopathology were vacAs2, one of the less virulent vacA alleles, and 66.6% of these patients with normal histopathology had cagA and no vacAs1 and cagE, which play an important role in virulence. In patients with active chronic gastritis, the vacAs1, vacAs2, cagA, and cagE virulence genes were found to be 44.4%, 51.8%, 33.3%, and 25.9%, respectively. While less virulent genes were detected in cases with normal histopathological evaluation, the positivity of genes known to be more virulent in active chronic gastritis cases was remarkable.

In countries where clarithromycin, metronidazole, and amoxicillin, which are the first-choice antibiotics, are used extensively in eradication treatment, the development of increasing resistance against these drugs causes treatment failure. Therefore, resistance determination has gained importance in the establishment of treatment protocols (20-22). Although many adult studies aimed at determining antibiotic resistance, there are not enough studies on children. Recent data on clarithromycin resistance in our country are generally obtained from studies conducted in adults, and resistance rates vary between 40.2% and 48% (23-25).The use of macrolide group antibiotics, especially in respiratory tract infections, is one of the most important reasons for the development of clarithromycin resistance. It is thought that clarithromycin resistance is a much more critical problem in developed countries, such as our country, where antibiotic use rates are high, or especially in developed countries where macrolide group antibiotics are prescribed for the treatment of respiratory tract infections (26-28).

In the study of Ozçay et al. (29), which was performed on 102 children diagnosed with H. pylori infection by urea breath test, serology, or culture, the rate of clarithromycin resistance was found as 18.1%.Karabiber et al. (10) reported that 51 (52%) of 98 PCR-positive children with H. pylori gastritis were culture-positive, and clarithromycin, metronidazole, and amoxicillin resistance were 23.5%, 11.7%, and 3.9%, respectively. In the same study, no relationship was found between the presence of cagA, and clarithromycin and metronidazole resistance. Clarithromycin resistance was found at a rate of 83.3% in cagE-positive patients, while this rate decreased to 16.7% in cagE-negative patients (10). In a study conducted on 118 patients, 52.7% of whom were children, in Spain in 2008, it was reported that the clarithromycin resistance measured by the E-test method was 35.6% (30).It was found that metronidazole and clarithromycin resistance tended to increase among children infected with H. pylori in China at rates of 49.2% and 34.9%, respectively (31). In Japan, resistance rates to metronidazole and clarithromycin were found to be 43.3% and 21.9%, respectively (32).Levofloxacin, which is effective against H. pylori in vitro, has more limited effects in vivo. Wong et al. (33) reported 18%,andPerna et al. (34) reported 30.3% levofloxacin-resistance. In another study, resistance to fluoroquinolone group drugs, which are not commonly used in children, was shown as 30.8% at the age of 0-6, and it was thought that resistance to these drugs was probably acquired from parents (35).The clarithromycin resistance observed in our study is compatible with studies in the literature. However, more meaningful results can be obtained by increasing the number of patients. If H. pylori cannot be destroyed sufficiently due to clarithromycin resistance, which is thought to be caused by unnecessary antibiotic use, triple and sequential treatments containing the fluoroquinolone group instead of the macrolide group are promising for older children. The rapid development of resistance to fluoroquinolone group antibiotics is worrying in this respect (35-38).

H. pylori is a microorganism with a high degree of genetic variability. While some studies reported a relationship between virulence genes and antibiotic resistance, some studies reported no such relationship (10, 39, 40). In our study, no relationship was found between vacA subtypes, cagA and cagE, and antibiotic resistance.

In the study conducted by Caliskan et al. (41) isolate a total of 98 H. pylori strains, 36 (36.7%) patients had clarithromycin resistance, 34 (35.5%) had metronidazole resistance, 29 (29.5%) had levofloxacin resistance, and multiple antibiotic resistance was detected in 19.3% of the patients. In the same study, the A2143G and A2144G genetic mutations were detected in 100% of clarithromycin-resistant strains. In our study, clarithromycin resistance was detected in 100% of the five patients who were found to be positive for the A2142G mutation and underwent antibiogram, and in 6 (55%) of the patients who were A2143G positive. Both mutations were found in male patients with levofloxacin resistance. Even at least one of these genetic mutations has the potential to become resistant to antibiotics.

A comparison of the vacAs1, vacAs2, cagA, and cagE virulence genes and A2142G and A2143G genetic mutations were made PCR-positive six patients with and seven parents of these patients. It has been shown that the H. pylori strains of the patient and their parents are different, and in 4 of the six patients we compared, the H. pylori strain with the same vacA allele as their parents was detected, whereas bacteria with different cagE and cagA genes were detected in these patients. It is known that the cagA and cagE genes located on the Cag Pathogenicity Island (cagPAI) of H. pylori may be acquired later or that existing genes may be lost in the future. While evaluating the differences in H. pylori strains between patients and parents, it should be kept in mind that there may be changes in cagPAI in the future. In addition, children can be infected with more than one strain of H. pylori at the same time. By increasing the number of biopsy samples, the possibility of detecting other strains of H. pylori can be increased.

Since the PCR-RFLP method, which examines the 1162 bp specific region of the vacA gene in our study to detect point mutations effective in clarithromycin resistance, is a sensitive method that gives a very heterogeneous band profile, as shown in other studies, it has been concluded that H. pylori isolation in gastric biopsy samples taken from pediatric patients is not a suitable method for determining kinship transmission (42).

CONCLUSION

In conclusion, H. pylori infection was positive in approximately one-third of the children with dyspeptic complaints. It was observed that these patients mostly had the vacAs2 genotype. Even individuals with at least one of the genetic mutations A2142G and A2143G genetic mutations have the potential to be resistant to antibiotics. A high rate of resistance has been found against clarithromycin used in the standard triple therapy for H. pylori in children in our country. H. pylori infection eradication rates are also low with standard triple therapy in clinical studies. Therefore, it is appropriate to develop new treatment modalities for treating H. pylori infection in children.

References

World Health Organization. A joint statement on tourism and COVID-19—UNWTO and WHO call for responsibility and coordination. Published, February 27, 2020. Accessed, September, 2021. Available from:https://www.who.int/news/item/27-02-2020-a-joint-statement-on-tourism-and-covid-19---unwto-and-who-call-for-responsibility-and-coordination.

World Health Organization. COVID-19: rights, roles and responsibilities of health workers, including key considerations for occupational safety and health. Published, 2019; (December):1-3. Accessed, September, 2021. Available from:https://www.who.int/docs/default-source/coronaviruse/who-rights-roles-respon-hw-covid-19.pdf?sfvrsn=bcabd401_0

On being human in the face of a pandemic. Nat Cancer. 2020; 1: 371.

Horton R. Offline: COVID-19 is not a pandemic. Lancet. 2020; 396: 874.

Gokdemir O, Pak H, Bakola M, Bhattacharya S, Hoedebecke K, Jelastopulu E. Family Physicians’ Knowledge about and Attitudes towards COVID-19 - A Cross-sectional Multicentric Study. Infect Chemother. 2020; 52: 539-49.

Rawaf S, Allen LN, Stigler FL, Krignos D, Yamamto HQ, Weel CV, et al. Lessons on the COVID-19 pandemic, for and by primary care professionals worldwide. Eur J Gen Pract. 2020; 26: 129-33.

Krist AH, DeVoe JE, Cheng A, Ehrlich T, Jones SM. Redesigning Primary Care to Address the COVID-19 Pandemic in the Midst of the Pandemic. Ann Fam Med. 2020; 18: 349-54.

Lee JQ, Loke W, Ng QX. The Role of Family Physicians in a Pandemic: A Blueprint. Healthcare (Basel). 2020; 8: 198.

Kearon J, Risdon C. The Role of Primary Care in a Pandemic: Reflections During the COVID-19 Pandemic in Canada. J Prim Care Community Health. 2020; 11: 2150132720962871.

McAuley A, Stewart B, Siemens G, Cormier D. The MOOC model for digital practice. University of Prince Edward Island; 2010.

Christensen G, Steinmetz A, Alcorn B, Bennett A, Woods D, Emanuel EJ. The MOOC Phenomenon: Who Takes Massive Open Online Courses and Why? SSRN Electron J. 2013.

Hoedebecke K, Mahmoud M, Yakubu K, Kendir C, D’Addosio R, Maria B, et al. Collaborative global health E-learning: A Massive Open Online Course experience of young family doctors. J Family Med Prim Care. 2018; 7: 884-7.

Breslow L, Pritchard D, DeBoer J, Stump G, Ho AD, Seaton DT. Studying learning in the worldwide classroom: Research into edX’s first MOOC.2013.

Braun V, Clarke V. Using thematic analysis in psychology. Qual Res Psychol. 2006; 3: 77-101.

Carter N, Bryant-Lukosius D, Dicenso A, Blythe J, Neville AJ. The use of triangulation in qualitative research. Oncol Nurs Forum. 2014; 41: 545-7.

Kern DE, Wright SM, Carrese JA, Jr ML, Simmons JM, Novack DH, et al. Personal growth in medical faculty: A qualitative study. West J Med. 2001; 175: 92-8.

Duffy FD, Holmboe ES. Self-assessment in lifelong learning and improving performance in practice: Physician know thyself. J Am Med Assoc. 2006; 296: 1137-9.

Yıldırım A, Şimşek H. Sosyal Bilimlerde Nitel Araştırma Yöntemleri. 2011.

Patton MQ. Nitel araştırma ve değerlendirme yöntemleri. 3. baskı. Bütün M, Demir SB, (Translation editors). Pegem Akademi; 2014. p. 1-598.

Camargo CP, Tempski PZ, Busnardo FF, Martins MA, Gemperli R. Online learning and COVID-19: a meta-synthesis analysis. Clinics (Sao Paulo). 2020; 75: e2286.

Elzainy A, El Sadik A, Al Abdulmonem W. Experience of e-learning and online assessment during the COVID-19 pandemic at the College of Medicine, Qassim University. J Taibah Univ Med Sci. 2020; 15: 456-62.

Goodyear-Smith F, Kinder K, Eden AR, Strydom S, Bazemore A, Philips R, et al. Primary care perspectives on pandemic politics. Glob Public Health. 2021; 16: 1304-19.

Pathogenic and Genetic Characteristics of Helicobacter Pylori, and its Relationship with Drug-Resistance