Evaluation of the helicobacter pylori, resistance and virulance factors by molecular methods in patients with dyspepsia

Clarithromycin resistance in H.pylori is considered an important cause for treatment failure. We aimed to detect H. pylori and determine clarithromycin resistance which is due to A2142G (A2146G), A2142C (A2146C), A2143G (A2147G), A2143C and A2144G point mutations in 23S rRNA gene by fluorescence in situ hybridization (FISH), real-time PCR, conventional culture and antimicrobial susceptibility testing and also to identify cagA positivity and vacA genotypes. Two-hundred-thirty-four patients with dyspepsia (65M, 169F; mean age 43.8±14.0 years) admitted to Gastroenterology Outpatient Clinic at Dokuz Eylül University Hospital were taken into the study. The diagnosis of H. pylori infection was defined as positive for at least two positive results with rapid urease test, histopathology and culture which were applied to antrum and corpus biopsy specimens. H. pylori cagA positive PCR products ranging 370-570 bp were amplified. H. pylori vacA genotypes PCR products; 290 bp for m1 region, 350 bp for m2 region, 176 bp for s1 region, 200 bp for s2 region were amplified. Formalin fixed paraffin embedded 468 antrum and corpus tissue sections were applied FISH (BACTfish, Hungary) (2143 and 2144 positions in 23SrRNA gene) method. DNA of 468 antrum and corpus biopsy specimens were extracted by QIAamp DNA mini kit and Real-time PCR (A2142G,A2142C,A2143G three point mutations in 23SrRNA gene) methods were applied to them. E-test was used to assess clarithromycin susceptibility in the isolated H. pylori strains. One-hundred-sixty-four (70.1%) patients were H. pylori positive according to at least two positivity. One-hundred-fourteen (69.5%) of 164 patients were culture positive, and 137 (83.5%) were positive by FISH, 157 (95.7%) were positive by real-time PCR. The sensitivity of FISH (83.5%) was significantly better, but not the specificity (91.4%) for detection of H. pylori, when compared with culture sensitivity (69.5%) and specificity (100%). The sensitivity and specificity of real-time PCR was 95.7%, 70%, respectively. Among H. pylori-positive patients, FISH detected clarithromycin resistance in 20.2%, E-test in 28.0%, real-time PCR in 34.4% of samples considering both antrum and corpus. The concordance between E-test and FISH was 89.5%. The concordance between E-test and real-time PCR was 75.4%. Among H. pylori-culture positive patients; 53 (46.5%) were cagA positive and no correlation was found between cagA positivity and clarithromycin resistance. m1s1 genotypes of vacA is in 29 (25.4%) patients (21 cagA+); m1s2 genotypes in 1 (0.9%) patient; m2s2 genotypes in 40 (35.1%) patients (4 cagA+) ; m2s1 genotypes in 35 (30.7%) patients (25 cagA+) were detected. Nine patients have mixed genotype strains. No relationship between vacA genotypes and clarithromycin susceptibility were found. As expected, vacA m1s1 and m2s1 were associated with CagA positivity and vacA m2s2 was associated with CagA negativity. As a conclusion, FISH significantly increases the sensitivity to detect H. pylori in the clinical microbiology laboratory when compare with traditional culture techniques. FISH technique is a reliable and highly sensitive method, especially useful when a quick decision is necessary for the management of diagnosis and treatment for patient with dyspepsia.