Prevalence of Helicobacter pylori cagA and vacA genes in Cypriot patients

Introduction: The prevalence of H. pylori varies with geographic locations. To date there are no epidemiological data on its prevalence in Cyprus; therefore, we determined the prevalence and molecular characteristics of H. pylori infection in Cypriot patients. Methodology: DNA extracted from 103 gastric biopsies was analyzed for the presence of H. pylori by PCR using primers for ureA. H. pyloripositive biopsies were characterized by PCR using specific primers for cagA and vacA genes. The presence of clarithromycin-associated resistant mutations such as A2143G, A2142G, A2142C in 23S rRNA gene of H. pylori-positive patients was determined using a real-time PCR allelic discrimination assay. Results: H. pylori was detected in 41 (39.8%) biopsies and, out of these, 17 (41.5%) tested positive for the cagA gene. The vacA alleles m1, m2, s1a, s1b, and s2 were detected in 7 (17.1%), 34 (82.9%), 12 (29.3%), 2 (4.9%), and 22 (53.7%) isolates, respectively. One (2.4%) biopsy was vacA s1a and s2-positive while one (2.4%) was positive for vacA s1a, s1b, and s2. Three (7.3%) biopsies were untypable for vacA s1, s1b, and s2. The majority (35; 85.4%) of strains were susceptible to clarithromycin while two (4.9%) had the A2143G mutation. Three (7.3%) had a mixture of an A2143G point mutant and susceptible strains while one (2.4%) had a mixture of an A2142G point mutant and susceptible strains. Conclusions: The distribution of the virulence factors cagA and vacA in the Cypriot strains resembled that of strains circulating in Middle Eastern countries geographically close to Cyprus.

Standard treatment for H. pylori infection involves a triple therapy with proton pump inhibitors, amoxicillin, and clarithromycin [21].The prevalence of clarithromycin resistance varies between 2% and 25% among different geographic areas but generally has been increasing worldwide [22].More than 90% of clarithromycin-resistant cases have been attributed to the three main 23S rRNA point mutations (A2143G, A2142G, A2142C of domain V), with different prevalence rates of these mutations reported worldwide [23][24][25][26].
In Cyprus, there is no data regarding the prevalence and molecular characteristics of H. pylori in patients with gastrointestinal discomfort.This is due to the fact that research in Cyprus has just started to grow in the last ten years.The proposed study, which was the first of this type conducted in Cyprus, aimed to determine (a) the prevalence of H. pylori infection, (b) the cagA and vacA genotypes of H. pylori isolates, (c) the prevalence of clarithromycin-associated resistant mutations in Cypriot patients, and (d) the effect of diet on H. pylori-associated symptoms.

Patients
The study included 103 adult patients who underwent routine upper gastrointestinal endoscopy due to dyspeptic complaint at Apollonion Hospital, Nicosia, Cyprus.At endoscopy, an antral/lower body biopsy was collected from each patient.Each gastric biopsy was subjected to PCR and real-time PCR analysis.None of the enrolled patients received antimicrobial therapy prior to collection of the gastric biopsy sample.This study was approved by the Cyprus national bioethics committee.All patients provided informed consent to participate in the study.

Diet regiment program
Following the collection of a gastric biopsy sample, all enrolled patients agreed by signing an informed consent form to participate in a diet regiment program aiming to evaluate the effect of diet in alleviating the gastrointestinal symptoms experienced on the day of gastrointestinal endoscopy.Initially patients underwent a full anthropometric assessment (weight, height, Body Mass Index, fat percentage) and filled out a specifically designed food frequency questionnaire.According to the results of the anthropometric assessment and the food frequency choices, each patient received a specific diet regiment.Briefly, each diet regiment suggested having meals at regular times each day and was divided into multiple small meals throughout the day.Specific recommendations such as which foods to avoid were also given at the beginning of the diet.A monthly follow-up session was scheduled and attended by all patients, during which their progress with regard to the symptoms and overall dietary status was assessed.This study was approved by the Cyprus national bioethics committee.
PCR detection of ureA, cagA, and vacA in gastric biopsies DNA was isolated from fresh gastric biopsies (25mg) using a QIAamp DNA blood mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.Isolated DNA was stored at -20 o C until analysis.The DNA concentration was determined with a spectrophotometer and ranged from 19.5 ng/L to 26.8 ng/L.The genes ureA, cagA, vacA midregion (m1 and m2) and signal region (s1a, s1b, s2) were amplified by polymerase chain reaction (PCR).PCR was performed in a 25 L reaction volume containing 1x PCR buffer supplemented with 1.5 mM MgCl 2 (Applied biosystems, Foster City, CA, USA), 200 M of each deoxynucleotide triphosphate, sense and antisense primers at concentrations ranging from 0.3 M-1 M, 1.5 U of AmpliTaq Gold DNA polymerase (Applied biosystems, Foster City, CA, USA), and 1 L aliquot of extracted DNA.The primers used and their details are shown in Table 1.The amplification reactions were performed using an MJR PTN-200 PCR machine (MJ Research Inc, Watertown, USA).The PCR conditions were as follows: (A) ureA: Initial denaturation step for 10 minutes at 95°C, followed by 37 cycles of 30 seconds at 95°C, 45 seconds at 45°C, and 72 °C for 30 seconds; (B) cagA: initial denaturation of 10 minutes at 95°C, followed by 37 cycles of 30 seconds at 95°C, 45 seconds at 55°C, and 72°C for 30 seconds; (C) vacA m1/m2/s1a/s1b/s2: initial denaturation of 10 minutes at 94°C, followed by 35 cycles of 45 seconds at 94°C, 45 seconds at 57°C, and 72°C for 45 seconds.PCR products were separated by electrophoresis on a 2% agarose gel and stained with ethidium bromide.

Real-time PCR detection of mutations associated with clarythromycin resistance
The A2142G, A2142C, A2143G point mutations in 23S rRNA gene were analyzed using a TaqMan real-time PCR allelic discrimination assay (Applied Biosystems, Foster city, USA) as previously described [27].Assays were prepared to enable scoring of both genotypes in a single reaction (wild type and mutation for each of the three mutation sites).Probes and primers were designed to hybridize with wild type and mutant DNA.Because a previous study showed that the conjugation of minor groove binder (MGB) to oligonucleotides stabilizes nucleic acid duplexes, causing a dramatic increase in oligonucleotide melting temperature (Tm), we used attachment of the MGB which enables the use of shorter fluorogenic probes, thus resulting in improved mismatch discrimination [28].The probes used were distinguished by being labeled with different fluorescent reporter dyes (FAM dye and VIC dye).A substantial increase in FAM or VIC fluorescence indicated homozygosity for the FAM-or VIC-specific allele, while an increase in both signals indicated heterozygosity [29].Details of the primers and probes used are shown in Table 2. Briefly, the real-time PCR mixture was prepared in a 25-L reaction volume containing Universal PCR master mix (Applied Biosystems, Foster City, CA, USA), 0.2 M of each primer, 0.1 M of VIC-labelled TaqMan MGB probe (for wild type), 0.1 M of FAM-labelled TaqMan MGB probe (for mutant), and 1 L aliquot of extracted DNA.Real-time PCR analysis was performed with an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA) and the conditions of the PCR amplification were as follows: 95 o C for 10 minutes and 40 cycles of 92 o C for 15 seconds and 60 o C for 1 minute.All samples were detected in duplicate and positive and negative controls were included in each assay.Following a successful realtime PCR, data were analyzed with the use of Sequence Detector Software (SDS) (Applied Biosystems, Foster city, USA).Allelic discrimination results were analyzed and depicted in the form of scatter plots of wild type versus mutant allele.Scatter plots consisted of three major clusters that varied along the horizontal axis (wild type homozygous), vertical axis (mutated type homozygous), or diagonal axis (heterozygous).

Detection of H. pylori infection and characterization of H. pylori positive strains in terms of cagA and vacA
Gastric biopsies from all 103 patients included in the study were initially assessed for the presence of H. pylori by PCR using ureA-specific primers encoding the urease gene.The 411-bp PCR product indicating the presence of H. pylori was detected in 41 patients (39.8%), whereas 62 patients (60.2%) were negative for the same PCR product.All H. pylori-positive biopsies were further analyzed to determine their cagA and vacA status.Seventeen biopsies (41.5%) were positive for the cagA gene, with the remaining 24 (58.5%) were classified as cagA-negative.In parallel, we characterized the vacA gene and allele types (Table 3).The primers used allowed amplification and differentiation of type s1a, s1b, and s2 vacA alleles and type m1 and m2 vacA alleles.The vacA midregion was detected in all H. pylori strains studied.In detail, the vacA m1 allele was found in 7 (17.1%)strains and the vacA m2 allele in 34 strains (82.9%).For the signal-region, in strains where a single vacA allele was found, the majority (22; 53.7%) were

Prevalence of clarithromycin-resistant H. pylori strains
Clarithromycin resistance was assessed using a TaqMan real-time PCR allelic discrimination assay.Analysis included all biopsies obtained that tested positive for H. pylori before the patient underwent any treatment.The results related to clarithromycinresistant H. pylori strains are shown in Table 4. Overall, 35 (85.4%) biopsies contained wild type H. pylori strains.The remaining 6 (14.6%) biopsies were determined to have either resistant or a mixture of resistant and wild type H. pylori strains.In detail, two (4.9%) biopsies were infected exclusively with resistant strains harboring the A2143G mutation, whereas the remaining four (9.7%) cases were characterized by a mixture of resistant and wild type strains.Three (7.3%) of these cases harbored wild type and resistant strains with the A2143G mutations, whereas one (2.4%)case harbored wild type and resistant strains with the A2142G mutation.

cagA status, vacA alleles and clinical diagnosis of H. pylori positive patients
According to the literature, virulent cytotoxin vacAand cagA-producing strains are found more frequently among patients with peptic ulcer and gastric cancer [5][6][7]30].In addition, in other studies, patients with various clinical symptoms including gastritis,  5.

Effect of diet on H. pylori-associated symptoms
The effect of diet on H-pylori-associated symptoms was assessed two months following the initiation of a diet regiment program tailored to the needs of each patient.Results analysis revealed that most of the symptoms were minimized in all patients irrespective of their H. pylori status, especially the feeling of fullness in the upper abdomen, dyspepsia, nausea, epigastric pain, subcternal distress, and regurgitation.Patients who succeeded in losing weight (as instructed by the clinical dietician), as well as improving their diet, reported alleviation of abdominal pain and better intestinal function.When the anthropometric assessment determined that weight loss was indicated as part of the treatment, a 5% loss of weight was found to be sufficient to improve abdominal bloating and gastrointestinal discomfort.Also, eating dairy products in moderation (no more than three servings per day) and avoiding entirely particular fruits and vegetables reduced symptoms of gastritis.In conclusion, our results indicate that the implemented diets did not have a direct effect on H. pylori infection but nevertheless they contributed to the improvement of the overall health status of the patients.

Discussion
Several studies have highlighted that the prevalence of H. pylori infection as well as the incidence and/or severity of H. pylori-related gastroduodenal diseases may be different among geographic areas.This can be partly attributed to the different distribution of H. pylori virulence factors around the world [34].Table 6 summarizes the distribution of the main H. pylori virulence factors worldwide.The present study is the first to determine the prevalence of H. pylori infection in Cypriot patients and molecularly characterize the two main H. pylori virulence-associated genes vacA and cagA.The predominant single genotype in the 41 biopsies found positive for H. pylori by PCR was the vacA m genotype, with 34 (82.9%)being vacA m2 positive and 7 (17.1%)vacA m1 positive, followed by the vacA s genotype, with 24 (58.5%) and 16 (39%) being positive for the vacA s2 and vacA s1 alleles, respectively.The cagA gene was amplified in 17 (41.5%) of the biopsies.This result was different from previously reported prevalence of cagA (66-73%) in Europe but in agreement with the majority of studies conducted in Middle Eastern countries (i.e., Turkey, Egypt, Israel and Jordan) where the cagA genotype was reported to vary between 26% and 44% [35][36][37][38][39].In a similar manner, the prevalence rates of the vacA m2 (82.9%), m1 (17.1%), and s2 (58.5%) genotypes detected in Cyprus were in agreement with those reported in other Middle Eastern countries (Table 6) [35][36][37][38][39] rather than those reported for Europe [12,13,[40][41][42].The similarities in terms of the genotypes and their prevalence identified in the present study and what has been reported in Middle Eastern countries could be attributed to a geographic influence important in the adaptation of H. pylori to the environment and climate conditions.The close resemblance of strains in neighboring countries was also reported in Israel, Egypt, and Jordan [37][38][39], as well as in Germany, France, and Poland [42,43].The prevalence of the vacA s1 allele has been reported to range between 46% and100% in the Middle East [35], 48% and 89% in Europe [12,13,[40][41][42], and 57% and 68% in America [14,44,45].In the present study, the vacA s1 allele was detected in 16 (39%) H. pyloripositive biopsies.
Clarithromycin resistance in H. pylori mainly results from point mutations in the peptidyltransferase loop region of the 23S rRNA gene [46].In the present study, the most frequent point mutation was A2143G in 12.2% of Cypriot strains, followed by the A2142G mutation detected in 2.4% of cases.These results were in agreement with those of other studies in Europe, the Middle East, and America, showing that the A2143G mutation is the one most frequently detected in H. pylori strains, followed by the A2142G mutation [22,[47][48][49] [14,44,45] Evaluation of the molecular characteristics of H. pylori strains in conjunction with clinical diagnosis revealed that most gastritis cases were characterized by a vacA m2 genotype.The presence of the cagA and vacA s1a allele in 5/5 (100%) of the peptic ulcer disease cases is in agreement with results obtained in previous studies showing an association between these virulence factors and peptic ulcer development [5,6,12,35,[50][51][52].Furthermore, as previously reported [14,20], the cagA gene was mostly associated with the vacA s1a allele (10/17; 58.8%), compared to vacA s1b and s2 alleles that were found in 2/17 (11.8%) and 4/17 (23.5%) of cagA-positive strains.
As part of the study, all enrolled patients, H. pylori positive and H. pylori negative, were given diets tailored to their needs in an attempt to alleviate their symptoms.Overall, symptoms were improved in all patients, with symptom improvement being more evident in some patients compared to others.The results highlight the need for a comprehensive approach toward managing these groups of patients in Cyprus.The described study takes a first step in that direction by providing information on the molecular characteristics of infecting strains that can assist physicians in taking the appropriate steps for patient treatment and care.

Conclusion
The present study has provided information that was previously unavailable regarding the prevalence and molecular characteristics of H. pylori infection in Cypriot patients.It would be of interest to continue the described study on a larger group of patients and to evaluate in further studies the molecular epidemiology of H. pylori infection in the general population.

Table 1 .
Target gene, primer name, oligonucleotide primers and expected product size positive for the vacA s2 allele, whereas 12 (29.3%)and 2 (4.9%) H. pylori strains were respectively positive for the vacA s1a and s1b alleles.In a minority of cases H. pylori positive biopsies were positive for more than one vacA signal region alleles, with one (2.4%)classified as positive for the vacA s1a/s2 genotype and one (2.4%)being positive for all three vacA signal region alleles tested.Three (7.3%) H. pylori strains were untypable for the the vacA signal region alleles tested.The most frequent vacA allelic combination in the H. pylori strains examined

Table 2 .
Sequences of primers and probes for detection of mutations associated with clarythromycin resistance

Table 5 .
Distribution of cagA and vacA alleles in patient groups GERD: gastroesophageal reflux disease, PUD: peptic ulcer disease

Table 6 .
Distribution of main H. pylori virulence factors in Europe, Middle East, and America