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Comparison of Culture With Antibiogram to Next-Generation Sequencing Using Bacterial Isolates and Formalin-Fixed, Paraffin-Embedded Gastric Biopsies

      Background & Aims

      The decline in Helicobacter pylori cure rates emphasizes the need for readily available methods to determine antimicrobial susceptibility. Our aim was to compare targeted next-generation sequencing (NGS) and culture-based H pylori susceptibility testing using clinical isolates and paired formalin-fixed, paraffin-embedded (FFPE) gastric biopsies.

      Methods

      H pylori isolates and FFPE tissues were tested for susceptibility to amoxicillin, clarithromycin, metronidazole, levofloxacin, tetracycline, and rifabutin using agar dilution and NGS targeted to 23S rRNA, gyrA, 16S rRNA, pbp1, rpoB and rdxA. Agreement was quantified using κ statistics.

      Results

      Paired comparisons included 170 isolates and FFPE tissue for amoxicillin, clarithromycin, metronidazole, and rifabutin and 57 isolates and FFPE tissue for levofloxacin and tetracycline. Agreement between agar dilution and NGS from culture isolates was very good for clarithromycin (κ = 0.90012), good for levofloxacin (κ = 0.78161) and fair for metronidazole (κ = 0.55880), and amoxicillin (κ = 0.21400). Only 1 isolate was resistant to tetracycline (culture) and 1 to rifabutin (NGS). Comparison of NGS from tissue blocks and agar dilution from isolates from the same stomachs demonstrated good accuracy to predict resistance for clarithromycin (94.1%), amoxicillin (95.9%), metronidazole (77%), levofloxacin (87.7%), and tetracycline (98.2%). Lack of resistance precluded comparisons for tetracycline and rifabutin.

      Conclusions

      Compared with agar dilution, NGS reliably determined resistance to clarithromycin, levofloxacin, rifabutin, and tetracycline from clinical isolates and formalin-fixed gastric tissue. Consistency was fair for metronidazole and amoxicillin. Culture-based testing can predict treatment outcomes with clarithromycin and levofloxacin. Studies are needed to compare the relative ability of both methods to predict treatment outcomes for other antibiotics.

      Keywords

      Abbreviations used in this paper:

      FFPE (formalin-fixed, paraffin-embedded), MIC (minimal inhibitory concentration), NGS (next-generation sequencing), NPV (negative predictive value), PCR (polymerase chain reaction), PPI (proton pump inhibitor), PPV (positive predictive value)
      See Covering the Cover synopsis on page 1351; See editorial on page 1367.

       Background and Context

      Cure rates using empiric therapy for Helicobacter pylori infections have declined steadily, stimulating a need for a widely available method for susceptibility testing. Next-generation sequencing (NGS) is a candidate to replace or supplement culture, as it can use isolates, biopsies, formalin-fixed biopsies, or stools to rapidly and reliably identify alterations in the genetic sequence of microbial genes resulting in antimicrobial resistance.

       New Findings

      Compared to agar dilution, NGS reliably determined resistance to clarithromycin, levofloxacin, rifabutin, and tetracycline from clinical isolates and formalin-fixed gastric tissue. Consistency was fair for metronidazole and amoxicillin.

       Limitations

      Limitations include lack of sufficient isolates resistant to rifabutin and tetracycline and inability to correlate the susceptibility profiles to the outcomes of therapy.

       Impact

      Molecular-based susceptibility testing has the potential to replace culture-based testing.
      Infectious disease therapies have traditionally been susceptibility-based, with treatment outcomes being defined by cure rates. Helicobacter pylori is an outlier, as most often therapy is prescribed without reference to local susceptibility data and the results are often suboptimal.
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      In infectious disease therapy, empiric therapy is used when the prevalence of susceptible organisms in the population is known to be high and high cure rates can be expected, or when therapy is given before the results of culture become available, with the understanding that the therapy will be revised (eg, to a more narrow-spectrum antibiotic) when the microbiology results become available.
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      The general unavailability of culture-based susceptibility testing for H pylori has resulted in the almost universal reliance on hopeful (empiric) therapy and a high proportion of treatment failures.
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      Unsatisfactory treatment outcomes coupled with increasing worldwide antimicrobial resistance have resulted in a movement toward the adoption of the general principles of antimicrobial therapy
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      Graham DY, Liou JM. Primer for development of guidelines for Helicobacter pylori therapy using antibiotic stewardship [published online ahead of print March 26, 2021. Clin Gastroenterol Hepatol https://doi.org/10.1016/j.cgh.2021.03.026

      and of antimicrobial stewardship, which is based on the use of optimized susceptibility-based therapies designed to reliably achieve high cure rates while simultaneously minimizing the development of resistance.
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      ,
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      Traditional culture-based susceptibility testing requires access to gastric contents and although minimally invasive approaches for collection of gastric juice, mucus, or tissue using tubes, strings, brushes, or forceps have been developed, testing is rarely done. Successful culture-based testing also requires appropriate biopsy handling, special transport conditions, and an experienced laboratory, and, even when this system works effectively, results are rarely available within 2 weeks. Molecular-based testing is being used increasingly to confirm the presence of an H pylori infection, as well as to identify alterations in the genetic sequence of microbial genes resulting in antimicrobial resistance.
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      Tshibangu-Kabamba E, Yamaoka Y. Helicobacter pylori infection and antibiotic resistance—from biology to clinical implications [published online ahead of print May 17, 2021. Nat Rev Gastroenterol Hepatol 2021;18:613-629.

      Modern molecular testing is rapid, with a turnaround time of less than 1 week and can provide susceptibility results for multiple antibiotics.
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      Molecular-based Helicobacter pylori susceptibility testing is almost ready for prime time.
      Testing can use gastric contents (ie, gastric juice, mucus, or mucosal biopsies), formalin-fixed, paraffin-embedded (FFPE) tissue, feces, or repurposing of tissue taken for histology or rapid urease tests.
      Until recently, the majority of molecular testing was designed to identify single or small sets of mutations in specific genes or in limited regions to detect resistance to clarithromycin or fluoroquinolones, either singly or simultaneously.
      • Redondo J.J.
      • Keller P.M.
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      A novel RT-PCR for the detection of Helicobacter pylori and identification of clarithromycin resistance mediated by mutations in the 23S rRNA gene.
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      However, H pylori has enormous genetic diversity, with high rates of mutation and recombination events, such that the mechanisms of antimicrobial resistance are both heterogeneous and intricate. For example, mutations or DNA sequence changes associated with metronidazole resistances have been described in multiple genes and regions, including the rdxA, frxA, fdxB, and recA genes, as well as nucleotide alterations within genes or regions, such as nonsense, missense, frameshift mutations, deletions, and insertions.
      • Jenks P.J.
      • Edwards D.I.
      Metronidazole resistance in Helicobacter pylori.
      • van der Wouden E.J.
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      • Kusters J.G.
      • et al.
      Mechanism and clinical significance of metronidazole resistance in Helicobacter pylori.
      • Hu Y.
      • Zhang M.
      • Lu B.
      • et al.
      Helicobacter pylori and antibiotic resistance, a continuing and intractable problem.
      Because of this complexity, polymerase chain reaction (PCR)-based tests using hybridization or enzymatic analytic methods are often not feasible for simultaneously testing of multiple antibiotics or for reliably detecting resistance to metronidazole. Sanger sequencing provides a relatively accessible approach and has been used for resistance analysis, but the method is limited by low throughput and lacks the sensitivity to detect mutations/variances with frequencies <10%–20%.
      • Chin E.L.H.
      • da Silva C.
      • Hegde M.
      Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations.
      Most commercially available kits for molecular testing for H pylori resistance are designed for use with culture isolates or fresh samples. In contrast, next-generation sequencing (NGS) technology can be used for the detection of mutation/DNA variances in a wide variety of indications, such as cancer precision medicine, genetic tests, and infectious diseases. Molecular testing has become increasing available and affordable, with its use being enhanced by technical developments regarding error correction strategies in NGS to allow accurate identification of low-level genetic variations.
      • Salk J.J.
      • Schmitt M.W.
      • Loeb L.A.
      Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations.
      For example, NGS is now used successfully in drug susceptibility profiling for Mycobacterium tuberculosis.
      • Ransom E.M.
      • Potter R.F.
      • Dantas G.
      • et al.
      Genomic prediction of antimicrobial resistance: ready or not, here it comes!.
      This study compared an NGS method (PyloriAR NGS, developed by American Molecular Laboratories, Vernon Hills, IL) for the detection of antimicrobial resistance markers from 2 different sources of H pylori: H pylori isolates cultured from gastric biopsies and DNA extracted from FFPE tissue blocks containing gastric mucosal biopsies. The molecular results were compared with a gold standard—susceptibility testing by agar dilution—and included the following 6 antimicrobial drugs: amoxicillin, clarithromycin, levofloxacin, metronidazole, tetracycline, and rifabutin.

      Methods

      The goal of this study was to compare susceptibility results obtained by culture and agar dilution with molecular-based analysis of H pylori antimicrobial resistance markers using isolates and tissue blocks of gastric mucosal biopsies. Gastric biopsy specimens were obtained from the antrum and corpus, respectively, from treatment-naïve adults (aged 18–70 years) who participated in a nationwide US H pylori eradication study clinical trial.
      • Graham D.Y.
      • Canaan Y.
      • Maher J.
      • et al.
      Rifabutin-based triple therapy (RHB-105) for Helicobacter pylori eradication: a double-blind, randomized, controlled trial.
      In all subjects, the presence of H pylori infection was originally determined by 13C urea breath tests, and confirmed by upper endoscopy with positive culture, histology, or rapid urease or Campylobacter-like organism test.

       Study Design

      Cases with both positive cultures and matching paraffin-embedded gastric biopsies were included. Before sending, H pylori isolates were re-randomized at Texas Children’s Hospital. Paraffin-embedded gastric mucosal biopsies were coded separately at the Miraca Life Sciences Laboratory. Both sets were sent for molecular testing and after culture and molecular assays, the data sets were unlocked and the results were compared. The following issues were addressed: how well the NGS predicted susceptibility corresponded to the culture-based susceptibility results from the same isolates; and how well NGS predicted susceptibility obtained from formalin-fixed gastric tissue corresponded to H pylori culture–based susceptibility data obtained from the same individuals. As antral and corpus samples were combined, all results are reported per patient not in relation to individual biopsy sites.

       Samples

       Antimicrobial susceptibility testing

      Minimal inhibitory concentrations (MICs) for amoxicillin, clarithromycin, metronidazole, and rifabutin were obtained as part of the treatment study. Because the original clinical study did not include susceptibility testing for tetracycline or levofloxacin, available isolates that could be revived from frozen stocks and that grew in sufficient density for agar dilution testing were tested and included in this study. Clinical and Laboratory Standards Institute methods and guidelines were followed.
      Clinical and Laboratory Standards Institute
      Performance Standards for Antimicrobial Susceptibility Testing, 28th ed. CLSI supplement M100.
      ,
      Clinical and Laboratory Standards Institute
      Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 11th ed.
      The detailed methods were described previously.
      • Graham D.Y.
      • Canaan Y.
      • Maher J.
      • et al.
      Rifabutin-based triple therapy (RHB-105) for Helicobacter pylori eradication: a double-blind, randomized, controlled trial.
      ,
      • Hulten K.G.
      • Lamberth L.B.
      • Kalfus I.N.
      • et al.
      National and regional United States antibiotic resistance to Helicobacter pylori resistance to Helicobacter pylori. Lessons from a clinical trial.
      Antibiotics were obtained from US Pharmacopeia, North Bethesda, MD. H pylori ATCC 43504 was included as a control. Clinical and Laboratory Standards Institute and the European Committee on Antimicrobial Susceptibility Testing (www.eucast.org) interpretive break points were used to determine resistance rates.

       Gastric Biopsies

      Sets of biopsies (1 each from antrum and corpus) were obtained for rapid urease or Campylobacter-like organism test, histopathology (Miraca Life Sciences, Irving, TX [now Informed Diagnostics]) and culture and susceptibilities (Texas Children’s Hospital, Houston, TX) were published as part of a clinical trial comparing rifabutin triple therapy with proton pump inhibitor (PPI)-amoxicillin dual therapy.
      • Graham D.Y.
      • Canaan Y.
      • Maher J.
      • et al.
      Rifabutin-based triple therapy (RHB-105) for Helicobacter pylori eradication: a double-blind, randomized, controlled trial.

       Next-Generation Sequencing–Based Molecular Methods for Helicobacter pylori Antibiotic Resistance Analysis

      NGS was performed (PyloriAR; American Molecular Laboratories, Chicago, IL) to identify mutations or variances of the H pylori DNA present in fixed tissue and culture isolates. Markers of antibiotic resistance associated with 6 H pylori genes—23S rRNA, gyrA, 16S rRNA, pbp1, rpoB and rdxA—were used (Table 1).
      • Jenks P.J.
      • Edwards D.I.
      Metronidazole resistance in Helicobacter pylori.
      ,
      • Megraud F.
      H pylori antibiotic resistance: prevalence, importance, and advances in testing.
      • Owen R.J.
      Molecular testing for antibiotic resistance in Helicobacter pylori.
      • Versalovic J.
      • Shortridge D.
      • Kibler K.
      • et al.
      Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori.
      • Paul R.
      • Postius S.
      • Melchers K.
      • et al.
      Mutations of the Helicobacter pylori genes rdxA and pbp1 cause resistance against metronidazole and amoxicillin.
      • Gerrits M.M.
      • de Zoete M.R.
      • Arents N.L.
      • et al.
      16S rRNA mutation-mediated tetracycline resistance in Helicobacter pylori.
      • Okamoto T.
      • Yoshiyama H.
      • Nakazawa T.
      • et al.
      A change in PBP1 is involved in amoxicillin resistance of clinical isolates of Helicobacter pylori.
      Table 1Targeted Genes and Mutations Reported to Predict Antibiotic Resistance
      AntibioticsGenesMutations and positions
      Clarithromycin23S rRNAA2142G, A2143G, A2142C
      FluoroquinolonesgyrAA260T (Asn87Ile), T261A, T261G (Asn87Lys), G271A (Asp91Asn), G271T (Asp91Gly)
      MetronidazolerdxAInsertion/deletion causing new stop codon or frame shift
      Missense mutations: R16HC, M21R/T/V, C19Y, P51L, A67V, C87Y, C140Y, C184Y, G163D/S/V
      Amoxicillinpbp1Ser414Arg/Asn
      Tetracycline16S rRNATCT926-928AAG, T926A/C927A, T926A/T928G, T926C/C927A, T926C/T928G and C927A/T928G
      RifabutinrpoBMutation in codon 525 to 545
      The PyloriAR NGS method uses proprietary designs to enrich the targeted areas to ensure the coverage. This is especially important in FFPE samples that contain fragmented DNA. The NGS assay was validated and the major parameters were set as follows: correct identification of mutations or variances required a minimum of 500 properly mapped reads and a minimum of 25 variant reads. PylorAR NGS protocols including the following steps: DNA extraction, real-time PCR for H pylori confirmation and quantitation, NGS library preparation, sequencing, and data analysis, which are detailed below.

       Sample Processing and DNA Extraction

       DNA preparation of tissue samples

      Five 10-μm sections were cut from each FFPE tissue block (which contained both the antral and corpus biopsy) on a microtome. The DNA was extracted from the sections using a Qiagen QIAcube processor and FFPE tissue kits with a modified protocol. Briefly, tissue sections were deparaffinized and placed in a tube with lysis buffer. Proteinase K was added and they were incubated at 56°C for 60 minutes, followed by 90°C for an additional 60 minutes. The lysed samples were then mixed with buffer and DNA was extracted using an automated Qiagen QIAcube system. To evaluate the quality of the extracted DNA from FFPE tissue, a multiplex PCR qualification assay with the amplification of the human GAPDH gene encoding glyceraldehyde-3-phosphate dehydrogenase was developed and used. The multiplex PCR has a set of PCR primers capable of producing an amplicon ladder of 100, 200, 300, 400, and 500 base-pair fragments. The DNA recovered from FFPE gastric biopsy samples was considered good if the test produces 2–5 bands of the amplicon ladder and was acceptable for the NGS analysis.

       DNA preparation of H pylori isolates

      Bacterial pellets were harvested from fresh cultures of the combined antral and corpus biopsies on agar plates and resuspended in 100 μL sterile 0.9% NaCl in the Infectious Disease Research Laboratory at Texas Children’s Hospital. The culture samples were relabeled and sent in a blinded fashion to the molecular laboratory for NGS analysis. Frozen subcultures were sent to the molecular diagnostic laboratory on dry ice. For DNA extraction 75 μL of each sample was mixed with lysis buffer and proteinase K, incubated at 56°C for 30 minutes followed by incubation at 90°C for 5 minutes. The lysed samples were then loaded on the Qiagen QIACube for DNA extraction. The DNA extracted from tissue blocks and culture isolates was quantified with Qubit 2.0 (Thermo Fisher Scientific) and stored at 4°C temporarily for immediate downstream testing or stored at –20°C for analysis at a later time.

       Real-Time Quantitative Polymerase Chain Reaction for H pylori Confirmation and Quantitation

      To confirm the presence of and quantitate H pylori DNA from the tissue blocks and culture isolates, a multiplex real-time PCR was performed targeting the H pylori 23S rRNA gene. Human GAPDH gene was used as internal control for the tissue samples. PCR was carried out on QuantStudio 3 (Applied Biosystems) with the initial denaturation at 95°C for 3 minutes, followed by 40 cycles consisting of denaturation at 95°C for 3 seconds and annealing/extension at 60°C for 30 seconds. Data analysis was performed with the QuantStudio Design & Analysis Software, version 1.4.3, installed as part of the Applied Biosystems device. Eligible samples with sufficient DNA were moved to the next step for NGS library preparation.

       NGS library preparation

      The method uses a targeted and paired-end sequencing library preparation method. A multiplex PCR was then used to amplify the targeted areas from 6 genes of the H pylori genome, including 23S rRNA, gyrA, rdxA, pbp1, 16S rRNA, and rpoB, see Table 1. The obtained amplicons from each amplification reaction were purified with the Agencourt AMPure XP PCR Purification system following the manufacturer’s instructions. The adapters and indexes were added and then followed by multiple clean-up steps. The library prepared for H pylori NGS is subject to quality control with fragment analysis, which ensures the appropriate DNA fragments are generated. A representative set of library samples are run on Bioanalyzer 2100 (Agilent). For the H pylori library, the peak approximately 300 base pair is expected.

       Sequencing and Data Analysis

      The libraries were pooled and loaded on the MiSeq platform (Illumina, Inc) for sequencing. The FASTQ formatted sequence data were generated with the MiSeq Reporter Analysis Software after the sequencing run. The Q-score ≥30 of sequenced reads ranging from 85% to 93% was used to confirm the high quality of the sequencing runs and its acceptability for further analysis. The commercial software NextGENe (SoftGenetics) was used to analyze the sequence data to identify antibiotic resistance mutations. The FASTQ data were initially converted to a standard fasta format and low-quality reads were trimmed or removed. The remaining clean reads were aligned and mapped to corresponding genes from the reference H pylori 26695 genome sequence (GenBank accession NC_000915). In order to identify mutations or variances correctly and minimize sequencing errors, a minimum of 500 properly mapped reads were required for the detection of variance at a 5% frequency. NGS can also potentially identify mixed infections using the ratio of wild-type and resistant strains in the testing samples based on the variant frequency (see Supplementary Material). In this study, the samples were reported and counted as resistant when 5% or more mutations or variances associated with resistance were detected.

       Ethical Considerations

      The original study from which the samples were obtained was conducted in accordance with ethical principles of the Declaration of Helsinki and applicable regulatory requirements. The study protocol and all amendments were approved by the respective Institutional Review Boards of participating institutions. All patients provided written informed consent before participating in the study.
      • Graham D.Y.
      • Canaan Y.
      • Maher J.
      • et al.
      Rifabutin-based triple therapy (RHB-105) for Helicobacter pylori eradication: a double-blind, randomized, controlled trial.
      This molecular comparison study did not include patient data and only used deidentified tissue and bacterial isolate preparations, and was approved by the Institutional Review Board for Baylor College of Medicine and affiliated hospitals. All of the authors have had full access to all of the data.

       Statistical Analyses

      The comparisons between the results of the 2 of methods (molecular detection of resistance markers vs agar dilution [gold standard]) was based on the established breakpoints, which is referred to here as the MIC. The comparisons between the results of the 2 methods to predict reference/susceptibility were done using the statistical program at SciStat. Agreement was quantified by the κ statistic, where κ = 1 when there is perfect agreement between the classification systems; κ = 0 when there is no agreement better than chance; and κ is negative when agreement is worse than chance. The κ values with standard error and 95% confidence interval are provided.

      Results

       Cases in the Study

      Because of the double-blinded design of this study and the availability of tissue blocks and culture isolates, the number of isolates and tissue blocks for which paired comparisons could be done was less than the total number included in the clinical study. The presence of H pylori DNA was confirmed in all tissue blocks and isolates received by the molecular diagnostic laboratory using real-time PCR, and they were then processed further for NGS analysis. A total of 170 tissue blocks had paired isolates and corresponding MIC results for clarithromycin, metronidazole, amoxicillin, and rifabutin; and a subset of 57 tissue blocks had paired isolates and corresponding MIC results for fluoroquinolones and tetracycline. The sequences of antibiotic resistance-related genes or regions were retrieved from the annotated genomes to identify potential antibiotic-resistance mutations. Mutations in genes previously reported to be associated with clarithromycin, levofloxacin, metronidazole, amoxicillin, tetracycline, and rifampin resistance were examined and the correlation with the phenotypic results was determined. In all instances, at least 500 reads were reached by NGS for the gene regions and hot spot where the variants were called.

       Comparison of the Results of Culture Susceptibility (Minimal Inhibitory Concentration) and Resistance Marker Testing by Next-Generation Sequencing on Culture Isolates and Formalin-Fixed, Paraffin-Embedded Tissues

       Clarithromycin

      Clarithromycin resistance of H pylori has been related to 23S rRNA gene mutations. The most common mutation (approximately 90%) in clarithromycin-resistant strains is an adenine to guanine transition at position 2142 and 2143 and an adenine to cytosine transversion at position 2142.
      • Versalovic J.
      • Shortridge D.
      • Kibler K.
      • et al.
      Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori.
      There are 2 copies of 23S rRNA genes in the H pylori genome, but it was considered as resistant if the mutations were present in at least 1 copy. Comparison of the determination of resistance using MIC and PyloriAR NGS testing of frozen H pylori isolates (n = 170) had strong agreement (κ = 0.90012) with a significant correlation coefficient (P < .0001) (Table 2). The results comparing MIC and PyloriAR NGS molecular methods to test FFPE tissue (n = 170) also had a good agreement (κ = 0.81236) (P < .0001) (Table 3).
      Table 2The Accuracy of PyloriAR Next-Generation Sequencing in Predicating Helicoobacter pylori Antibiotic Resistance in Culture Isolates
      AntibioticsPyloriAR NGS culture isolateCulture (MIC)Sensitivity, %Specificity, %Predicative value, %Accuracy, %Agreement, κ coefficient
      (+)(–)PPVNPV
      Clarithromycin(+)283
       n = 170(–)213793.397.990.398.697.10.90012 (P < .0001)
       95% CI77.9–99.293.9–99. 675.2–96.694.7–99.693.3–990.8671–0.9253
      Amoxicillin(+)10
       n = 170(–)716212.5100.0100.095.995.90.21400 (P = .0051)
       95% CI0.3–52.697.7–100.094.7–96.891.7–98.30.0656–0.3531
      Metronidazole(+)689
       n = 170(–)296470.187.788.368.877.6%0.5588 (P < .0001)
       95% CI50–7977.9–94.280.2–93.461.7–75.270.63–83.70.4458–0.6543
      Rifabutin(+)01
       n = 170(–)016999.40.00100.0
       95% CI96.8–100
      Levofloxacin(+)314
       n = 57(–)22093.983.388.690.989.50.78161 (P < 0.0001)
       95% CI79.8–99.362.6–95.375.9–9572.1–97.578.5–960.61689–0.94633
      Tetracycline(+)00
       n = 57(–)1560.00100.098.298.2
       95% CI0.00–97.593.6–100.098.2–98.290.6–100
      (+), resistant; (–), sensitive; CI, confidence interval.
      Table 3The Accuracy of PyloriAR Next-Generation Sequencing in Determining the Antibiotic Resistance of Helicobacter pylori in Formalin-Fixed, Paraffin-Embedded Tissues
      AntibioticsPyloriAR NGS, FFPE resultsCulture (MIC)Sensitivity, %Specificity, %Predictive value, %Accuracy, %Agreement, κ coefficient
      (+)(–)PPVNPV
      Clarithromycin(+)288
       Total n = 170(–)213293.394.377.898.594.10.81236 (P < .0001)
       95% CI77.9–99.289–97.563.9–87.394.5–99.689.4–97.10.70076 to 0.92396
      Amoxicillin(+)10
       Total n = 170(–)716212.510010095.995.90.21400 (P = .0051)
       95% CI0.3–52.697.7–100.094.7–96.891.7–98.3–0.13849 to 0.56649
      Metronidazole(+)6810
       Total n = 170(–)296370.186.387.268.577.10.54645 (P < .0001)
       95% CI60–7976.2–93.279–92.561.2–74.970.0–83.10.42443 to 0.66847
      Rifabutin(+)00
       Total n = 170(–)0170100.0100.0
       95% CI97.8–100.0
      Levofloxacin(+)293
       Total n = 57(–)42187.987.590.684.087.70.74953 (P < .0001)
       95% CI71.8–96.667.6–97.376.9–96.667.4–9376.3–94.90.57597 to 0.92308
      Tetracycline(+)00
       Total n = 57(–)1560.0100.098.298.2
       95% CI0.0–97.593.6–100.098.2–98.290.6–100
      (+), resistant; (–), sensitive; CI, confidence interval.
      PyloriAR NGS results predicted H pylori resistance in isolates (susceptible or resistant) with a 97.1% accuracy compared to the gold standard (agar dilution) with a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 93.3%, 97.9%, 90.32%, and 98.6%, respectively. From the FFPE tissues, PyloriAR NGS predicted H pylori resistance in 94.1% of samples compared to the gold standard (MIC) with a sensitivity, specificity, PPV, and NPV of 93.3%, 94.3%, 77.9%, and 98.5%, respectively.

       Amoxicillin

      Amoxicillin resistance is still rare in H pylori but, when present, has primarily been linked to mutations in the gene pbp1 with a mutation at amino acid Ser414Arg/Asn. PyloriAR NGS applied Ser414Arg/Asn as the marker to predict resistance. Based on the κ statistic, the agreement between the isolates or FFPE tissue compared to the gold standard was fair but significant (both κ = 0.214) (P < .05). The accuracy of PyloriAR NGS in predicting H pylori resistance from culture isolates and FFPE tissue was the same: 95.9% (Tables 2 and 3). In addition to Ser414Arg/Asn mutation in the gene pbp1, other mutations, as shown in Supplemental Table 1, were observed in isolates and/or tissues; none were specifically associated with resistance. In 41 of the 170 tissue samples, D479E, T593A/K, and G595S were present; more often in susceptible than in resistant samples. In 170 isolates, D479E, T593A/K, and G595S mutations were present in 53 samples; more often in susceptible than in resistant samples (Supplementary Tables 2 and 3). Overall, compared to the gold standard, PyloriAR NGS was unable to confirm resistance in 7 of 170 isolates and paired 170 tissue samples. However, it is important to note and, as elaborated in the Discussion, in the clinical study from which the samples were obtained the agar dilution results often failed to reliably predict outcomes with the dual PPI-amoxicillin therapy.
      • Graham D.Y.
      • Canaan Y.
      • Maher J.
      • et al.
      Rifabutin-based triple therapy (RHB-105) for Helicobacter pylori eradication: a double-blind, randomized, controlled trial.

       Metronidazole

      Any mutation, insertion/deletion in gene rdxA resulting in a stop codon and amino acid frameshift can result in metronidazole resistance.
      • Moore J.M.
      • Salama N.R.
      Mutational analysis of metronidazole resistance in Helicobacter pylori.
      The mutation/DNA variances that have been associated with resistance include R16H, M21R/T/V, C19Y, P51L, A67V, C87Y, C140Y, C184Y, and G163D/S/V.
      • Paul R.
      • Postius S.
      • Melchers K.
      • et al.
      Mutations of the Helicobacter pylori genes rdxA and pbp1 cause resistance against metronidazole and amoxicillin.
      ,
      • Jenks P.J.
      • Ferrero R.L.
      • Labigne A.
      The role of the rdxA gene in the evolution of metronidazole resistance in Helicobacter pylori.
      ,
      • Chua E.-G.
      • Debowski A.W.
      • Webberley K.M.
      • et al.
      Analysis of core protein clusters identifies candidate variable sites conferring metronidazole resistance in Helicobacter pylori.
      The resistance results comparing agar dilution and PyloriAR NGS molecular testing with frozen H pylori isolates (n = 170) and the resistance results between agar dilution and PyloriAR NGS molecular testing with FFPE tissue (n = 170) had moderate agreement (κ = 0.55880 and κ = 0.54645, respectively) (P < .0001) (Tables 2 and 3). The predicted accuracy of PyloriAR NGS was 77.6% and 77% from culture isolates and FFPE tissues, respectively.

       Rifabutin

      Resistance of H pylori to rifabutin results from mutations in the rpoB gene.
      • Anjum M.F.
      • Zankari E.
      • Hasman H.
      Molecular methods for detection of antimicrobial resistance.
      There were no rifabutin-resistant cases among the 170 tissue samples by culture MIC and PyloriAR NGS. One sample was resistant by PyloriAR NGS molecular testing among the isolates, but was judged susceptible in the paired FFPE sample on the same subject. Based on these data, PyloriAR NGS molecular testing for predicting H pylori susceptibility to rifabutin achieved the specificity of 99.4% and 100% in culture isolates and FFPE tissues respectively (Tables 2 and 3). However, the lack of rifabutin resistance among the samples tested limits this analysis.

       Levofloxacin

      Resistance to levofloxacin results from mutations in the gyrA gene, most commonly caused by a point mutation at amino acid 87 (Asn to Lys or Ile) or 91 (Asp to Gly, Asn or Tyr).
      • Anjum M.F.
      • Zankari E.
      • Hasman H.
      Molecular methods for detection of antimicrobial resistance.
      There were 57 cases with FFPE tissue blocks and paired culture isolates with corresponding agar dilution results. There was a good agreement between agar dilution results and PyloriAR NGS of isolates, and between agar dilution results and PyloriAR NGS of FFPE tissues (κ = 0.78163 and κ = 0.74953, respectively) (P < .0001) (Tables 2 and 3).
      Compared to the gold standard, PyloriAR NGS of isolates identified H pylori resistance with an accuracy of 89.5% with sensitivity, specificity, PPV, and NPV of 93.9%, 83.3%, 88.6%, and 90.9%, respectively. PyloriAR NGS of FFPE tissue identified H pylori resistance with an accuracy of 87.7% with sensitivity, specificity PPV, and NPV of 87%, 87.5%, 90.6% and 84%, respectively.

       Tetracycline

      Tetracycline resistance has been associated with 3 nucleotide changes at position 926–928 in 16S rRNA gene of H pylori. If there is only 1 nucleotide change, the resistance level is low, whereas the triple mutation, such as AGA926-928TTC, results in high-level tetracycline resistance.
      • Gerrits M.M.
      • de Zoete M.R.
      • Arents N.L.
      • et al.
      16S rRNA mutation-mediated tetracycline resistance in Helicobacter pylori.
      ,
      • Gerrits M.M.
      • Berning M.
      • van Vliet A.H.
      • et al.
      Effects of 16S rRNA gene mutations on tetracycline resistance in Helicobacter pylori.
      NGS required at least 2 mutations in the 16S RNA gene to predict resistance to tetracycline. Because there are 2 copies of 16S rRNA genes in the H pylori genome, a sample was considered resistant if the mutations were present in either or both copies. PyloriAR NGS identified tetracycline resistance in in none of the 57 culture isolates and 1 of 57 (2.8%) isolates were resistant by agar dilution. Based on the data, the PyloriAR NGS predicted H pylori resistance to tetracycline with an accuracy of 98.2% in both isolates and FFPE tissues (Tables 2 and 3).

      Discussion

      With the exception of H pylori, antimicrobial therapy for infectious diseases is either directly or indirectly susceptibility-based, consistent with increasing pressure to uniformly use the principles of antimicrobial stewardship for managing infectious disease therapy.
      • Dyar O.J.
      • Huttner B.
      • Schouten J.
      • et al.
      What is antimicrobial stewardship?.
      The movement toward tailored or susceptibility-based H pylori therapies is also reflected in the more than 70 citations in PubMed,
      • Wenzhen Y.
      • Yumin L.
      • Quanlin G.
      • et al.
      Is antimicrobial susceptibility testing necessary before first-line treatment for Helicobacter pylori infection? Meta-analysis of randomized controlled trials.
      • Chen Y.
      • Liu Q.
      • Hu F.
      • et al.
      Identifying the best regimen for primary eradication of Helicobacter pylori: analysis of 240 cases.
      • Lopez-Gongora S.
      • Puig I.
      • Calvet X.
      • et al.
      Systematic review and meta-analysis: susceptibility-guided versus empirical antibiotic treatment for Helicobacter pylori infection.
      as well as editorials on this topic.
      • Graham D.Y.
      Editorial–Avoiding unethical Helicobacter pylori clinical trials: susceptibility-based studies and probiotics as adjuvants.
      In China, where H pylori antimicrobial resistance is especially prevalent, a number of studies have confirmed that using therapies based on an individual’s susceptibility test results provides highly effective outcomes, despite a high local prevalence of antimicrobial resistance.
      • Yu L.
      • Luo L.
      • Long X.
      • et al.
      Susceptibility-guided therapy for Helicobacter pylori infection treatment failures.
      • Chen Q.
      • Long X.
      • Ji Y.
      • et al.
      Randomised controlled trial: susceptibility-guided therapy versus empiric bismuth quadruple therapy for first-line Helicobacter pylori treatment.
      • Luo L.
      • Ji Y.
      • Yu L.
      • et al.
      14-day high-dose amoxicillin- and metronidazole-containing triple therapy with or without bismuth as first-line Helicobacter pylori treatment.
      • Luo L.
      • Huang Y.
      • Liang X.
      • et al.
      Susceptibility-guided therapy for Helicobacter pylori-infected penicillin-allergic patients: a prospective clinical trial of first-line and rescue therapies.
      The generally poor cure rates obtained with empiric use of the currently most popular triple therapies for H pylori infection reflect both the global increase in prevalence of antimicrobial resistance and the lack of availability of culture-based susceptibility testing.
      • Graham D.Y.
      • El-Serag H.B.
      European Registry on Helicobacter pylori management shows that gastroenterology has largely failed in its efforts to guide practitioners.
      ,
      • Nyssen O.P.
      • Bordin D.
      • Tepes B.
      • et al.
      European Registry on Helicobacter pylori management (Hp-EuReg): patterns and trends in first-line empirical eradication prescription and outcomes of 5 years and 21 533 patients.
      Molecular-based tests designed to identify antimicrobial resistance are now commercially available and theoretically could provide up-to-date local and regional H pylori susceptibility data to enhance cure rates and limit the development of antimicrobial resistance.
      • Leekha S.
      • Terrell C.L.
      • Edson R.S.
      General principles of antimicrobial therapy.
      ,
      Core elements of antibiotic stewardship. Centers for Disease Control and Prevention.
      ,
      • Dyar O.J.
      • Huttner B.
      • Schouten J.
      • et al.
      What is antimicrobial stewardship?.
      ,
      • Cambau E.
      • Allerheiligen V.
      • Coulon C.
      • et al.
      Evaluation of a new test, genotype HelicoDR, for molecular detection of antibiotic resistance in Helicobacter pylori.
      Available PCR-based tests are usually designed to detect 1 or a few individual targets.

      Tshibangu-Kabamba E, Yamaoka Y. Helicobacter pylori infection and antibiotic resistance—from biology to clinical implications [published online ahead of print May 17, 2021. Nat Rev Gastroenterol Hepatol 2021;18:613-629.

      In contrast, NGS methods can simultaneously identify DNA mutations or variances of H pylori genes responsible for resistance in all of the commonly used anti–H pylori antimicrobials.
      • Graham D.Y.
      Molecular-based Helicobacter pylori susceptibility testing is almost ready for prime time.
      ,
      • Saracino I.M.
      • Pavoni M.
      • Zullo A.
      • et al.
      Next generation sequencing for the prediction of the antibiotic resistance in Helicobacter pylori: a literature review.
      We used the PyloriAR NGS test (American Molecular Laboratories), which can simultaneously provide information regarding resistance in 6 commonly used anti–H pylori antimicrobials (Table 1). This method has advantages over whole genome sequencing, which, although it is an excellent research tool to identify new mutations, especially when using bacterial colonies, it is currently neither practical nor cost-effective for routine clinical use.
      • Chawla K.
      • Martinez E.
      • Kumar A.
      • et al.
      Whole-genome sequencing reveals genetic signature of bedaquiline resistance in a clinical isolate of Mycobacterium tuberculosis.
      ,
      • Jindal H.M.
      • Ramanathan B.
      • Le C.F.
      • et al.
      Comparative genomic analysis of ten clinical Streptococcus pneumoniae collected from a Malaysian hospital reveal 31 new unique drug-resistant SNPs using whole genome sequencing.
      The presence of fragmented DNA also makes whole genome sequencing more difficult when applied to FFPE tissue. The targeted sequencing approach using NGS is currently a more efficient and cost-effective approach compared to whole genome sequencing.
      PyloriAR NGS uses proprietary designs designed to enrich and cover multiple genes of interest and large regions that DNA mutation/variance that can cause resistance. This approach ensures sufficient sequencing coverage of FFPE tissues containing fragmented DNA or crosslinks between DNA and protein or other macromolecules. The advantages of this technology include the wide range of sample types that can be used, a high rate of successful sequencing (100% in this study) and rapid availability of results. Furthermore, for mixed samples containing both susceptible and resistant strains, the method can detect and predict the frequency of a mutation within the sample (see Supplementary Material, Supplementary Table 4). However, limitations do exist, including that it focuses on already known genomic regions for antimicrobial resistance, as the predictive value might be somewhat reduced because of DNA fragmentation of FFPE tissue samples.
      This study compared the results of culture-based and molecular-based susceptibility testing of 6 antibiotics using both FFPE biopsies and H pylori isolates obtained from the same subjects. Overall, molecular-based testing agreed with the MIC results obtained by agar dilution for clarithromycin and levofloxacin. The comparison between MIC and molecular results was less consistent for metronidazole, and the correlation between culture-based susceptibility testing and molecular testing with amoxicillin was relatively poor. Tetracycline and rifabutin resistance was rare to absent in our sample, which limited the evaluation. However, for susceptible isolates, both culture and molecular testing were highly concordant.
      Clarithromycin, levofloxacin, and, presumably, rifabutin resistance involves single targets that govern susceptibility vs resistance; clinically, resistance eliminates the antibiotic’s beneficial effects and reduces a triple therapy (eg, a PPI, clarithromycin, and amoxicillin) to PPI-amoxicillin dual therapy. Metronidazole is a prodrug that is activated intracellularly into a toxic version that destroys the bacterial DNA. Metronidazole susceptibility predicts treatment success, but the effect of resistance is not all or none, but depends on the composition of the therapy as well as the metronidazole dosage and duration of therapy. Amoxicillin resistance has been related to alterations in penicillin-binding proteins
      • Dore M.P.
      • Kwon D.H.
      • Sepulveda A.R.
      • et al.
      Stable amoxicillin resistance in Helicobacter pylori.
      ,
      • Rimbara E.
      • Noguchi N.
      • Kawai T.
      • et al.
      Mutations in penicillin-binding proteins 1, 2 and 3 are responsible for amoxicillin resistance in Helicobacter pylori.
      and is still rare among H pylori isolates from most regions. To date, no H pylori strains have been reported that produce β-lactamases. Our finding that culture-based amoxicillin susceptibility testing and molecular testing correlated poorly was also observed by of Azzaya et al.
      • Azzaya D.
      • Gantuya B.
      • Oyuntsetseg K.
      • et al.
      High antibiotic resistance of Helicobacter pylori and its associated novel gene mutations among the Mongolian population.
      The relative poor results obtained for amoxicillin and metronidazole are most likely the consequence of an insufficient molecular characterization of these antibiotic resistances. It is possible that genetic alterations in other genes could be associated with the increased MIC observed for these isolates. Our samples were derived from a study that included a PPI-amoxicillin dual therapy that potentially allowed the effect of amoxicillin resistance to be judged without the presence of a second antibiotic. Importantly, in that PPI-amoxicillin dual therapy study, MIC values for amoxicillin resistance did not reliably predict treatment failure or success.
      • Graham D.Y.
      • Canaan Y.
      • Maher J.
      • et al.
      Rifabutin-based triple therapy (RHB-105) for Helicobacter pylori eradication: a double-blind, randomized, controlled trial.
      However, outcomes assessment was complicated because the overall cure rate was low (57.7%; 95% confidence interval, 51.2%–64.0%), which was typical for studies of PPI-amoxicillin dual therapy in the United States.
      • Attumi T.A.
      • Graham D.Y.
      Increasing the duration of dual amoxicillin plus omeprazole Helicobacter pylori eradication to 6 weeks: a pilot study.
      • Attumi T.A.
      • Graham D.Y.
      High-dose extended-release lansoprazole (dexlansoprazole) and amoxicillin dual therapy for Helicobacter pylori infections.
      • Graham D.Y.
      • Javed S.U.
      • Keihanian S.
      • et al.
      Dual proton pump inhibitor plus amoxicillin as an empiric anti-H. pylori therapy: studies from the United States.
      • Laine L.
      • Johnson E.
      • Suchower L.
      • et al.
      US double-blind, controlled trials of omeprazole and amoxycillin for treatment of Helicobacter pylori.
      A pooled analysis of 5 randomized trials of amoxicillin-containing triple therapy showed that phenotypic amoxicillin resistance was associated with a decrease in cure rate.
      • Chen M.-J.
      • Wu M.-S.
      • Chen C.-C.
      • et al.
      Impact of amoxicillin resistance on the efficacy of amoxicillin-containing regimens for Helicobacter pylori eradication: analysis of five randomized trials.
      The majority of the published literature on molecular testing in H pylori has been concerned with detection of genotypic resistance unencumbered by comparisons with culture-based results or with clinical outcomes.
      • Domanovich-Asor T.
      • Motro Y.
      • Khalfin B.
      • et al.
      Genomic analysis of antimicrobial resistance genotype-to-phenotype agreement in Helicobacter pylori.
      Ultimately, the comparisons should include the response to treatment, which it often complicated by the use of multidrug regimens. Further studies are needed to identify which genes, or patterns of gene involvement, best correlate with outcome.
      In summary, the results of this study suggest that molecular methods can potentially augment or even replace the current in vitro methods for susceptibility testing, which are cumbersome, technically challenging, and time-consuming. As with other genomic-based methods, phenotypic, culture-based susceptibility testing and genomic prediction of resistance do not always correspond. Treatment failure is currently predicted by determining a breakpoint MIC value based on the results of clinical treatment data. Reliable predictive data for H pylori therapy are available for only clarithromycin and levofloxacin. Studies in populations with high prevalence of tetracycline, rifabutin, or amoxicillin resistance are needed to help clarify how to best use molecular testing results to reliable predict outcomes.

      Acknowledgments

      Drs. Hulten and Zhang contributed equally to the work. The authors thank Linda B. Lamberth, BS and Lauren M. Sommer, MS for performing the culture and susceptibility testing. The authors also thank Professor Maria Pina Dore, Sassari, Italy for pointing out that, based on the Latin derivation, the word empiric was being misused when associated with Helicobacter pylori therapy.

      CRediT Authorship Contributions

      David Y. Graham, MD (Conceptualization: Equal; Data curation: Equal; Formal analysis: Equal; Funding acquisition: Equal; Investigation: Equal; Methodology: Equal; Project administration: Equal; Supervision: Lead; Writing – original draft: Equal; Writing – review & editing: Equal). Kristina G. Hulten, PhD (Conceptualization: Equal; Data curation: Lead; Formal analysis: Equal; Investigation: Equal; Methodology: Equal; Project administration: Equal; Resources: Equal; Supervision: Lead; Writing – original draft: Equal; Writing – review & editing: Equal). Robert M. Genta, MD (Data curation: Equal; Formal analysis: Equal; Investigation: Lead; Resources: Equal; Supervision: Equal; Writing – original draft: Equal; Writing – review & editing: Equal). Ira N. Kalfus, MD (Conceptualization: Equal; Formal analysis: Equal; Funding acquisition: Equal; Investigation: Equal; Resources: Equal; Writing – original draft: Equal; Writing – review & editing: Equal). Yi Zhou, PhD (Data curation: Equal; Formal analysis: Equal; Investigation: Equal; Methodology: Equal; Resources: Equal; Software: Equal; Validation: Equal; Writing – original draft: Equal; Writing – review & editing: Equal). Hongjun Zhang, MD, PhD (Conceptualization: Equal; Data curation: Equal; Formal analysis: Lead; Funding acquisition: Equal; Investigation: Equal; Methodology: Lead; Project administration: Equal; Resources: Equal; Software: Lead; Supervision: Equal; Validation: Lead; Writing – original draft: Equal; Writing – review & editing: Equal).

      Supplementary Material

      Supplementary Table 1Amoxicillin Resistance Associated With Mutations in Pbp1 Reported in the Literature
      Mutations/positionsStudy first author
      This studyNishizawa
      • Nishizawa T.
      • Suzuki H.
      • Tsugawa H.
      • et al.
      Enhancement of amoxicillin resistance after unsuccessful Helicobacter pylori eradication.
      Gerrits
      • Gerrits M.M.
      • de Zoete M.R.
      • Arents N.L.
      • et al.
      16S rRNA mutation-mediated tetracycline resistance in Helicobacter pylori.
      Kim
      • Kim B.J.
      • Kim J.G.
      Substitutions in penicillin-binding protein 1 in amoxicillin-resistant Helicobacter pylori strains isolated from Korean patients.
      Kwon
      • Kwon Y.H.
      • Kim J.Y.
      • Kim N.
      • et al.
      Specific mutations of penicillin-binding protein 1A in 77 clinically acquired amoxicillin-resistant Helicobacter pylori strains in comparison with 77 amoxicillin-susceptible strains.
      Okamoto
      • Okamoto T.
      • Yoshiyama H.
      • Nakazawa T.
      • et al.
      A change in PBP1 is involved in amoxicillin resistance of clinical isolates of Helicobacter pylori.
      Paul
      • Paul R.
      • Postius S.
      • Melchers K.
      • et al.
      Mutations of the Helicobacter pylori genes rdxA and pbp1 cause resistance against metronidazole and amoxicillin.
      Rimbara
      • Rimbara E.
      • Noguchi N.
      • Kawai T.
      • et al.
      Mutations in penicillin-binding proteins 1, 2 and 3 are responsible for amoxicillin resistance in Helicobacter pylori.
      Tuan
      • Tuan V.P.
      • Narith D.
      • Tshibangu-Kabamba E.
      • et al.
      A next-generation sequencing-based approach to identify genetic determinants of antibiotic resistance in Cambodian Helicobacter pylori Clinical Isolates.
      V16IX
      V45IX
      A320VX
      F366LXX
      A369TX
      V374LXXX
      S402GX
      E406A/KXXX
      S414RXXXXXXX
      S417TX
      L423FX
      P473LX
      A474T/VX
      D479EX
      S467TX
      Y484CX
      N504DX
      T541IX
      S543RX
      N562YXXXX
      S589GXX
      G591KX
      T593A/G/K/PXXXXXXX
      G595SXX
      A599T/GXX
      P600TX
      V601GX
      Supplementary Table 2Mutations in the Gene pbp1 Detected With PyloriAR Next-Generation Sequencing in 170 Tissue Cases
      MutationsResistant by MICSusceptible by MIC
      S414R/N10
      F473L/V01
      D479E111
      T593A/K221
      G595S121
      Supplementary Table 3Mutations in the Gene pbp1 Detected With PyloriAR Next-Generation Sequencing in 170 Culture Isolates
      MutationsResistant by MICSusceptible by MIC
      S414R/N10
      F473L/V02
      D479E118
      T593A/K122
      G595S026
      Supplementary Table 4Mutation Frequency Changes Demonstrated by Next-Generation Sequencing Analysis With Mixing Culture Isolates
      Mixing testSample IDsNGS results, %
      23S rRNAgyrA
      1002A2143G 99.4A272G Asp91Gly 99.4
      1025NoneNone
      1002 + 1025 (mix)A2143G 47.1A272G Asp91Gly 42.3
      1645A2143G 6.2G271A Asp91Asn 99.8
      Test 11002 + 1025 + 1645 (mix)A2143G 25.2G271A Asp91Asn 49, A272G Asp91Gly 21
      1033A2143G 99.7None
      1036A2143G 99.8T261A Asn87LyS 98.7
      Test 21033 + 1036 (mix)A2143G 99.8T261A Asn87LyS 61.4
      1003A2143G 99.6A272G Asp91Gly 99.4
      1027NoneNone
      Test 31003 + 1027 (mix)A2143G 52.6A272G Asp91Gly 49.95

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