Gastric Juice-Based Real-Time PCR for Tailored Helicobacter Pylori Treatment: A Practical Approach

Abstract

A gastric juice-based real-time polymerase chain reaction (PCR) assay was established to identify Helicobacter pylori infection, clarithromycin susceptibility and human CYP2C19 genotypes and to guide the choice of proton pump inhibitor (PPI), clarithromycin and amoxicillin treatment for tailored H. pylori eradication therapy. From January 2013 to November 2014, 178 consecutive dyspeptic patients were enrolled for collection of gastric biopsy samples and gastric juice by endoscopy at the Peking University Third Hospital; 105 and 73 H. pylori-positive and -negative patients, respectively, were included in this study. H. pylori infection was defined as samples with both a strongly positive rapid urease test (RUT) and positive H. pylori histology. A series of primers and probes were distributed into four reactions for identifying the H. pylori cagH gene coupled with an internal control (Rnase P gene), A2142G and A2143G mutants of the H. pylori 23S rRNA gene, and single-nucleotide polymorphisms (SNPs) G681A of CYP2C19*2 and G636A of CYP2C19*3. The E-test and DNA sequencing were used to evaluate the H. pylori clarithromycin susceptibility phenotype and genotype. The SNPs CYP2C19*2 and CYP2C19*3 were also evaluated by nucleotide sequencing. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of this gastric juice-based real-time PCR assay were evaluated by comparing with the same measures obtained through gastric biopsy-based PCR and culture. The H. pylori diagnostic sensitivities of the culture, PCR, and gastric biopsy- and gastric juice-based real-time PCR assays were 90.48% (95/105), 92.38% (97/105), 97.14% (102/105) and 100% (105/105), respectively; the specificities of the above methods were all 100%. Higher false-negative rates were found among the gastric biopsy samples assessed by culture (10.48%, 11/105), PCR (7.62%, 8/105) and real-time PCR (2.86%, 3/105) than in gastric juice by real-time PCR. Regarding clarithromycin susceptibility, a concordance of 82.98% (78/94) and discordance of 17.02% (16/94) were observed among the different methods, discrepancies that mainly represent differences between the H. pylori clarithromycin susceptibility phenotype and genotype. Three coinfections of susceptible and resistant strains were detected, with resistant-to-susceptible ratios of 1.16, 3.44, and 8.26. The CYP2C19 genotyping results from gastric juice by real-time PCR were completely in accordance with those obtained from biopsy samples by conventional PCR. This gastric juice-based real-time PCR assay is a more accurate method for detecting H. pylori infection, clarithromycin susceptibility and CYP2C19 polymorphisms. The method may be employed to inform the choice of proton pump inhibitor (PPI), clarithromycin and amoxicillin treatment for tailored H. pylori eradication therapy.

What Is New in Helicobacter pylori Diagnosis. An Overview

“…In addition to gastric biopsies, molecular tests can be applied to the gastric mucus present on biopsy forceps placed into water or into the RUT gel [ 88 ], and the dual-priming oligonucleotide-based multiplex PCR, performed on CLO ® -test kits, proved to be superior to RUT and histology, in patients with a bleeding peptic ulcer [ 89 ]. Alternatively, molecular tests to detect H. pylori and its susceptibility to antibiotics can be performed on gastric juice [ 90 , 91 , 92 ]. A droplet-digital PCR may also be applied to formalin-fixed, paraffin-embedded gastric tissue to determine the presence of clarithromycin resistance [ 93 ], or by next generation sequencing to determine levofloxacin and tetracycline resistance [ 94 ] ( Figure 2 ).…”

Microarray-Based Detection and Clinical Evaluation for Helicobacter pylori Resistance to Clarithromycin or Levofloxacin and the Genotype of CYP2C19 in 1083 Patients

“…According to CYP2C19∗2 and CYP2C19∗3, the CYP2C19 phenotype has been classified into three groups: homozygous extensive metabolizers (Hom-EMs), heterozygous extensive metabolizers (Het-EMs), and poor metabolizers (PMs) [ 12 ]. Research shows that, in the first-line treatment based on PPI, PM patients can get the highest H. pylori eradication rate.…”

“…There are four most often observed point mutations associated with clarithromycin: A2144G, A2143G, A2142G, and A2143C in 23S rRNA [ 12 ]. “Hot-spots” (A2142G and A2143G, approximately 80% of all mutations) domain V has shown the major mechanism of clarithromycin resistance [ 11 , 13 ].…”

<p>Eradication Treatment of <em>Helicobacter pylori</em> Infection Based on Molecular Pathologic Antibiotic Resistance</p>

“…21 Three main alleles have been found, including CYP2C19*1 (wild-type), CYP2C19*2 (mutation in exon 5) and CYP2C19*3 (mutation in exon 4). 21,22 The sequences of primers were designed and synthesized for CYP2C19*2 (F: 5ʹ-AATTACAACCAGAGCTTGGC-3′ R: 5ʹ-TATCA CTTTCCATAAAAGCAAG-3′) and CYP2C 19*3 (F: 5ʹ-TATTATTATCTGTTA ACTAATATGA-3′ R: 5ʹ-ACTTCAGGGCTTGGTCAATA-3′). 21 They were detected by PCR amplification and the products were analyzed and sequenced at the NewLife Inc.…”