Comparison of the Diagnostic Performance of qPCR, Sanger Sequencing, and Whole-Genome Sequencing in Determining Clarithromycin and Levofloxacin Resistance in Helicobacter pylori

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.

Modern approach to the diagnosis of Helicobacter pylori infection

“…[35][36][37][38][39][40][41] The Genotype HelicoDR assay (Germany) and whole genome sequencing have been used for both clarithromycin and levofloxacin resistance. 42,43 Although there are a number of validated tests and test kits commercially available, the majority have been tested with gastric biopsies and they are infrequently performed. Overall, the availability of these tests and test kits has not changed the frequent admonition about the lack of susceptibility testing or the continuing decrease in cure rates.…”

Application of molecular techniques inHelicobacter pyloridetection: limitations and improvements

“…34 This will then aid the choice of an appropriate treatment strategy targeting strains with different susceptibility and virulence profiles. 30,34 Other molecular methods 25 to detect clarithromycin and levofloxacin resistance 35 36 and FISH 37,38 ; all of them showing higher specificity and simplicity than agar-dilution culture (E-test), the conventional antibiotic susceptibility test. 11 Another application of molecular tests is to study H. pylori evolution and human migration patterns via molecular phylogenetic analysis of 16S rRNA gene from different populations.…”

“…This will then aid the choice of an appropriate treatment strategy targeting strains with different susceptibility and virulence profiles 30,34 . Other molecular methods 25 to detect clarithromycin and levofloxacin resistance 35 in H . pylori include restriction fragment length polymorphism (RFLP), 3’‐mismatch PCR, whole genome sequencing, DNA chips, PCR line probe assay, amplification refractory mutation system‐based PCR (ARMS‐PCR), peptide nucleic acid (PNA)‐based PCR, dual‐priming oligonucleotide (DPO)‐based PCR 36 and FISH 37,38 ; all of them showing higher specificity and simplicity than agar‐dilution culture (E‐test), the conventional antibiotic susceptibility test 11 .…”

Development and Validation of Multiplex Quantitative PCR Assay for Detection of Helicobacter pylori and Mutations Conferring Resistance to Clarithromycin and Levofloxacin in Gastric Biopsy

“…65 In this study, the qPCR was able to detect the presence of mixed genotypes in H. pylori-negative biopsies by culture and mixed genotypes are still giving the bacterial load (10 1 bacterial cells for N87K and 10 5 bacterial cells for D91WT). A reasonable explanation could be that these mixed sequences are due to mixed infection 27 because there is a single-copy of gyrA gene presence in the H. pylori genome. 34 The presence of a mixed H. pylori population in the same sample detected by qPCR is an advantage when compared with culture.…”

“…21,[23][24][25] Conversely, there are only a few available qPCR assays for detection of H. pylori and mutations conferring resistance to both clarithromycin and levofloxacin antibiotics. 26,27 Therefore, the objective of this study was to develop and evaluate a multiplex qPCR assay for detection of H. pylori and gene mutations conferring clarithromycin and levofloxacin resistance in H. pylori in gastric biopsy specimens. This assay will provide us with a new tool along with rapid detection method and standard protocols for management and monitoring, and in improving outcomes of H. pylori eradication therapy.…”