Abstract
Helicobacter pylori antibiotic resistance is increasing worldwide, emphasizing the urgent need for more rapid resistance detection prior to the administration of H. pylori eradication regimens. Macrolides and fluoroquinolones are widely used to treat H. pylori. In this study, we aimed to compare the diagnostic performance of A) 23SrDNA qPCR (with melting curve analysis) and an in-house developed gyrA qPCR followed by Sanger sequencing with a commercial IVD-marked hybridization probe assay (for 23SrDNA and gyrA) using 142 gastric biopsies (skipping culturing) and B) the same two qPCR for 23SrDNA and gyrA (including Sanger sequencing) with whole-genome sequencing (WGS) and phenotypic characterization of clarithromycin and levofloxacin resistance using 76 cultured isolates. The sensitivity of both qPCRs was 100% compared to that of the commercial IVD-marked hybridization probe assay for the detection of H. pylori in gastric biopsies (without resistance testing). The specificity of the qPCR gyrA followed by Sanger sequencing was 100%, indicating that the best sequence identity was always H. pylori. The results show good agreement between molecular tests, especially between qPCR (inclusive Sanger sequencing) and WGS. Discrepancies (concerning mutated or wild type of positive H. pylori gastric biopsies) were observed between Sanger sequencing of the gyrA gene and the corresponding commercial hybridization probe assay, mostly because the high sequence diversity of the gyrA gene even at positions adjacent to the relevant codons of 87 and 91 interfered with obtaining correct results from the hybridization probe assay. Interestingly, we found several mixed sequences, indicating mixed populations in the gastric biopsies (direct detection without culturing). There was a high percentage of both levofloxacin and clarithromycin resistance in gastric biopsies (both between 22% and 29%, direct detection in gastric biopsies). Therefore, we recommend analyzing both targets in parallel. We confirmed that phenotypic resistance is highly correlated with the associated mutations. We concluded that the two qPCR followed by Sanger sequencing of the gyrA gene is a fast, cost-effective and comprehensive method for resistance testing of H. pylori directly in gastric biopsies.
Highlights
Helicobacter pylori is a gram-negative bacterium that infects approximately half of the human population worldwide
DNA from 142 gastric biopsy specimens (104 positive and 38 negative samples) was analyzed by gyrA quantitative realtime PCR assays (qPCR) followed by Sanger sequencing, and the Genotype HelicoDR showed a concordance of 100% regarding positive and negative results
With qPCR followed by Sanger sequencing, 23 specimens with amino acid exchange leading to resistance were detected
Summary
Helicobacter pylori is a gram-negative bacterium that infects approximately half of the human population worldwide. An increasing trend of clarithromycin resistance in H. pylori can be observed worldwide and in European countries, with an overall primary clarithromycin resistance rate of 17%– 21% (de Francesco et al, 2010; Dolak et al, 2017; Bilgilier et al, 2018). Levofloxacin resistance has not been studied as extensively, there is a trend towards increasing primary and secondary levofloxacin resistance in H. pylori (Hug and Rossi, 2001; Mandell et al, 2007). With rising antibiotic resistance rates, the usefulness of the currently applied “test-and-treat” strategy is questionable, emphasizing the need for rapid and accurate diagnostics to assess drug resistance in H. pylori prior to the administration of antibiotics
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