Determination of clarithromycin and levofloxacin susceptibility by molecular methods in Helicobacter pylori genomic DNA isolated from gastric biopsy and stool specimens

Clarithromycin and levofloxacin resistance are the most important cause for treatment failure of Helicobacter pylori infection. We aimed to assess the efficacy of GenoType HelicoDR method and its applicability for stool specimens to detect H. pylori and determine clarithromycin and levofloxacin susceptibilities, simultaneously besides the phenotypic susceptibility testing and molecular methods. Fifty-eight patients with dyspepsia (40F, 18M; 41.1 ± 13.5 years) were studied. All tests used in this study were performed to both antrum and corpus biopsy specimens and stool specimens of these patients. H.pylori positivity was defined according to at least two positivity of culture, histopathology and rapid urease test. Clarithromycin and levofloxacin susceptibilities were determined by E-test as a traditional method. We modified GenoType HelicoDR test for stool specimens as a non-invasive molecular method to detect H. pylori and to determine clarithromycin and levofloxacin susceptibilities. GenoType HelicoDR test was also performed to gastric biopsy specimens. Furthermore, FRET-real-time PCR was performed to both biopsy and stool specimens for melecular detection of H. pylori and determination of clarithromycin susceptibility. Biopsy and colony DNA specimens of the patients which had discrepant clarithromycin and levofloxacin susceptibility results among used different methods were sent for sequencing. Fifty-one (87.9%) patients were H.pylori positive. Fourty-nine (84.5%) patients were culture positive. Thirty-five (71.4%), 35 (71.4%) patients were susceptible; 10 (20.4%), 9 (18.4%) were resistant; 4 (8.2%), 5 (10.2%) patients' antrum and corpus presented a double population of susceptible and resistant bacteria by E-test for clarithromycin and levofloxacin, respectively. All patients were H. pylori positive by GenoType HelicoDR; 37 (63.8%), 33 (56.9%) were susceptible; 5 (8.6%), 11 (19%) were resistant; 16 (27.6%), 14 (24.1%) presented a double population for clarithromycin and levofloxacin, respectively. GenoType HelicoDR test was adapted successfully for stool specimens. Fifty-four (98.2%) patients were positive and 1 (1.8%) patient was negative for H. pylori by stool Genotype HelicoDR. Stool GenoType HelicoDR results of 35 (63.6%) and 40 (72.7%) patients were correlated with the gastric biopsy results of GenoType HelicoDR for clarithromycin and levofloxacin, respectively. The sensitivity of stool GenoType HelicoDR was 100% (κ = 0.225). Correlation between FRET-real-time PCR and GenoType HelicoDR methods to determine clarithromycin susceptibility was 81.0 % and 66.7 % in gastric biopsy and stool specimens, respectively. Three new mutations (C2136T, C2310A, C2428T) were determined in three patients' sequence analyses conferring wild type for known main three mutations associated with clarithromycin resistance. E-test results of these patients were also resistant for clarithromycin. GenoType HelicoDR and FRET-real-time PCR methods are rapid and culture independent tests with the ability to study directly from biopsy specimens and also has capability for detection of mixed infections including both wild type and resistant H. pylori strains. However, molecular determination of levofloxacin susceptibility simultaneously with claritromycin susceptibility is the best advantage of GenoType HelicoDR test especially in the region which is clarithromycin resistance is high. As a non-invasive molecular method, ability of using stool GenoType HelicoDR is very valuable in clinical setting providing to determine antimicrobial susceptibility prior to the treatment without invasive sampling.