Cloning, expression and characterization of recombinant CagA protein of Helicobacter pylori using monoclonal antibodies: Its potential in diagnostics.

Helicobacter pylori CagA protein plays an important role in the severity of the gastric diseases. Our aims were to clone the cagA 5'- conserved region of the gene, characterize the recombinant CagA (rCagA) protein by monoclonal antibodies (mAbs) and to use this protein for the detection of anti-CagA antibodies by an ELISA test. Our developed rCagA protein (67 kDa) showed an amino acid sequence homology of 83% and 80% with Western and East Asian type strains respectively. Two anti-rCagA (BS-53, CK-02) mAbs and 2 additional rCagA proteins of smaller sizes (60 kDa, 28 kDa) were developed for epitope determination. The BS-53 mAb recognized all 3 rCagA proteins while CK-02 mAb recognized only 2 of them indicating recognition of different epitopes. An in-house indirect ELISA using rCagA was developed to detect anti-CagA antibodies in sera of 59 patients. The ELISA results obtained when compared to those of the PCR gave a sensitivity, specificity and accuracy of 81%, 100% and 88% respectively. We have developed for the first time: a rCagA protein that showed high sequence homology with both Western and East Asian type strains and an indirect ELISA of high performance which can be used to detect anti-CagA antibodies in sera of infected patients worldwide.

Antimicrobial resistance of Helicobacter pylori strains to five antibiotics, including levofloxacin, in Northwestern Turkey.

INTRODUCTION: Antibiotic resistance is the main factor that affects the efficacy of current therapeutic regimens against Helicobacter pylori. This study aimed to determine the rates of resistance to efficacy clarithromycin, amoxicillin, tetracycline, levofloxacin and metronidazole among H. pylori strains isolated from Turkish patients with dyspepsia. METHODS: H. pylori was cultured from corpus and antrum biopsies that were collected from patients with dyspeptic symptoms, and the antimicrobial susceptibility of H. pylori was determined using the E-test (clarithromycin, amoxicillin, tetracycline, metronidazole and levofloxacin) according to the EUCAST breakpoints. Point mutations in the 23S rRNA gene of clarithromycin-resistant strains were investigated using real-time PCR. RESULTS: A total of 98 H. pylori strains were isolated, all of which were susceptible to amoxicillin and tetracycline. Of these strains, 36.7% (36/98) were resistant to clarithromycin, 35.5% (34/98) were resistant to metronidazole, and 29.5% (29/98) were resistant to levofloxacin. Multiple resistance was detected in 19.3% of the isolates. The A2143G and A2144G point mutations in the 23S rRNA-encoding gene were found in all 36 (100%) of the clarithromycin-resistant strains. Additionally, the levofloxacin MIC values increased to 32 mg/L in our H. pylori strains. Finally, among the clarithromycin-resistant strains, 27.2% were resistant to levofloxacin, and 45.4% were resistant to metronidazole. CONCLUSIONS: We conclude that treatment failure after clarithromycin- or levofloxacin-based triple therapy is not surprising and that metronidazole is not a reliable agent for the eradication of H. pylori infection in Turkey.

Antimicrobial resistance of Helicobacter pylori strains to five antibiotics, including levofloxacin, in Northwestern Turkey

INTRODUCTION: Antibiotic resistance is the main factor that affects the efficacy of current therapeutic regimens against Helicobacter pylori. This study aimed to determine the rates of resistance to efficacy clarithromycin, amoxicillin, tetracycline, levofloxacin and metronidazole among H. pylori strains isolated from Turkish patients with dyspepsia. METHODS: H. pylori was cultured from corpus and antrum biopsies that were collected from patients with dyspeptic symptoms, and the antimicrobial susceptibility of H. pylori was determined using the E-test (clarithromycin, amoxicillin, tetracycline, metronidazole and levofloxacin) according to the EUCAST breakpoints. Point mutations in the 23S rRNA gene of clarithromycin-resistant strains were investigated using real-time PCR. RESULTS: A total of 98 H. pylori strains were isolated, all of which were susceptible to amoxicillin and tetracycline. Of these strains, 36.7% (36/98) were resistant to clarithromycin, 35.5% (34/98) were resistant to metronidazole, and 29.5% (29/98) were resistant to levofloxacin. Multiple resistance was detected in 19.3% of the isolates. The A2143G and A2144G point mutations in the 23S rRNA-encoding gene were found in all 36 (100%) of the clarithromycin-resistant strains. Additionally, the levofloxacin MIC values increased to 32 mg/L in our H. pylori strains. Finally, among the clarithromycin-resistant strains, 27.2% were resistant to levofloxacin, and 45.4% were resistant to metronidazole. CONCLUSIONS: We conclude that treatment failure after clarithromycin- or levofloxacin-based triple therapy is not surprising and that metronidazole is not a reliable agent for the eradication of H. pylori infection in Turkey. .

Evaluation of the effect of cagPAI genes of Helicobacter pylori on AGS epithelial cell morphology and IL-8 secretion.

Helicobacter pylori cagPAI genes play an important role in pathogenesis, however little is known about their functions in isolates from Turkish patients. We aimed to evaluate the intactness and the effect of the cagPAI genes (cagT, cagM, cagE, cagA) and cagA EPIYA motifs on the AGS morphological changes and IL-8 induction. Of 53 patients 38 were found infected with H. pylori. PCR amplification of the cagPAI genes showed 42.1 % intact, 39.5 % partially deleted and 18.4 % with complete deletions. Isolates from gastritis, duodenal and gastric ulcer patients with intact and partially deleted cagPAI genes induced higher IL-8 secretion than those with complete deletions. Isolates from gastritis patients had higher deletion frequencies of the cagT and cagM genes than the other two genes. Infection of AGS cells with isolates that possess intact cagPAI and EPIYA-ABC resulted in the formation of the hummingbird phenotype. The cagA positive isolates induced higher IL-8 secretion than cagA negative isolates. Isolates from DU patients with more than one EPIYA-C motif induced higher concentrations of IL-8 than those with EPIYA-ABC. In conclusion, the intactness of the cagPAI in our isolates from different patients was not conserved. An intact cagPAI was found to play an important role in the pathogenesis of DU but not GU or gastritis. The cagA gene, but not other cagPAI genes, was associated with the induction of IL-8 and the morphological changes of the AGS cells. An increase in the number of EPIYA-C motifs had noticeable effect on the formation of the hummingbird phenotype.

Phylogenetic analysis of Helicobacter pylori cagA gene of Turkish isolates and the association with gastric pathology.

BACKGROUND: The cagA gene is one of the important virulence factors of Helicobacter pylori. The diversity of cagA 5' conserved region is thought to reflect the phylogenetic relationships between different H. pylori isolates and their association with peptic ulceration. Significant geographical differences among isolates have been reported. The aim of this study is to compare Turkish H. pylori isolates with isolates from different geographical locations and to correlate the association with peptic ulceration. METHODS: Total of 52 isolates of which 19 were Turkish and 33 from other geographic locations were studied. Gastric antral biopsies collected from 19 Turkish patients (Gastritis = 12, ulcer = 7) were used to amplify the cagA 5' region by PCR then followed by DNA sequencing. RESULTS: The phylogenetic tree displayed 3 groups: A) a mix of 2 sub-groups "Asian" and "African/Anatolian/Asian/European", B) "Anatolian/European" and C) "American-Indian". Turkish H. pylori isolates clustered in the mixed sub-group A were mostly from gastritis patients while those clustered in group B were from peptic ulcer patients. A phylogenetic tree constructed for our Turkish isolates detected distinctive features among those from gastritis and ulcer patients. We have found that 2/3 of the gastritis isolates were clustered alone while 1/3 was clustered together with the ulcer isolates. Several amino acids were found to be shared between the later groups but not with the first group of gastritis. CONCLUSIONS: This study provided an additional insight into the profile of our cagA gene which implies a relationship in geographic locations of the isolates.

DNA sequence analysis of cagA 3' motifs of Helicobacter pylori strains from patients with peptic ulcer diseases.

The Helicobacter pylori cagA gene is a major virulence factor that plays an important role in gastric pathologies. DNA sequence data for the cagA 3' region of Western isolates differ markedly in their EPIYA motifs from those of East Asian isolates. An increase in the number of these motifs is known to be associated with gastric cancer. Whether such an association is also the case for peptic ulceration was investigated in this study. Gastric biopsies were collected from 96 patients with duodenal ulcer (DU), gastric ulcer (GU) and gastritis. The types of EPIYA motif detected by PCR among 28 DU strains were 13 ABC, eight ABCC, six ABCCC, and in one patient both ABC and ABCCCCC; among nine GU strains were two ABC, five ABCC and two ABCCC; and among 40 gastritis strains were 35 ABC and five ABCC. DNA sequencing was carried out to confirm the detection of the EPIYA motif types and to analyse their peptide sequences. A significant association was found between the number of the EPIYA-C motifs (>or=2) and peptic ulceration (P=0.00001) compared with gastritis. In conclusion, this study shows that our patients harboured cagA-positive H. pylori strains with EPIYA motifs of the Western type and that the increase in the number of EPIYA-C motifs was significantly associated with DU and GU but not with gastritis, indicating predictive association with the severity of the disease.

Genotyping of Helicobacter pylori strains from gastric biopsies by multiplex polymerase chain reaction. How advantageous is it?

Recent application of multiplex polymerase chain reaction (PCR) for genotyping Helicobacter pylori direct from biopsies revealed variable results (detection of amplicons from DNA extracted by boiling biopsies, variable size amplicons and deletions, uniform intensity of amplicon bands). We aimed to look at how applicable the technique is for determining cagA and vacA genotypes and to correlate the results with the severity of the disease. H. pylori strains from 52 patients (35 duodenal ulcers [DUs], 7 gastric ulcers [GUs], 10 gastritis) were included. Three antral biopsies were obtained for Campylobacter-like organism (CLO) and PCR. Primers for cagA, vacA s1s2, and m1m2 alleles were used. No PCR amplicons were detected from boiling biopsies; thus, DNA was extracted by QIAamp kit. H. pylori was positive in 84.6% of the patients (85.7% DU, 100% GU, and 70% gastritis). The cagA gene was detected in 86.6% DU, 71.4% GU, and 57.0% gastritis patients. The vacA allelic distribution among cagA-positive strains was 80.7% s1m1 in DU and 60.0% in GU patients, whereas 75.0% of gastritis had s1m2. No variability in the amplicon sizes was found, and the intensity of the amplicon bands was not uniform. A deleted band of approximately 420 bp below the m1 band was detected in strains from 2 DU and 1 GU patients. Although the multiplex PCR is a rapid and an effective tool for detecting several genes in a single-step system, one has to adjust for optimization of the technique when genotyping H. pylori direct from biopsies. A significant association was found between the cagA-positive vacA-s1m1 genotype and peptic ulcers.