Effect of the CYP2C19 Polymorphism in Helicobacter Pylori Eradication
Effect of the CYP2C19 Polymorphism in Helicobacter Pylori Eradication
Background: Triple therapy efficacy against Helicobacter pylori is low worldwide, thus
 alternatives must be sought to improve eradication. Aim: To determine CYP2C19 genetic
 polymorphism effect on H. pylori eradication.

 Methods: A randomized single blinded clinical trial including 133 patients was carried-out.
 H. pylori infection was confirmed by histology and microbiological test. Antibiotic
 susceptibility to amoxicillin and clarithromycin was performed to avoid confusion bias in
 analysis results. CYP2C19 polymorphism "asterisk" *1, "asterisk"*2 and "asterisk" *3 was
 analyzed by Real time PCR (Roche ®), and nested PCR for CYP2C19 "asterisk" *17 polymorphism.
 Participants were randomized into two groups for different H. pylori therapies, one with
 standard omeprazole doses and another with omeprazole doses depending on CYP2C19
 polymorphism. H. pylori eradicating was verified by stool antigen testing (Meridian ®). The
 general results was analysis by statistical computer program and the effectiveness of each
 therapy was analyzed by intention to treat (ITT) and by protocol (PP).

 The study allowed to know the prevalence of the main polymorphisms of CYP2C19 in
 Bogotá-Colombia, also allowed to know the effectivenesses of the two therapies evaluated for
 H. pylori infection. Additionally, the importance of personalized medicine in H. pylori
 eradication therapy was known.
Introduction:

 H. pylori has been declared and ratified as carcinogen I by the World health organization
 (WHO) International agency for research on cancer (IARC). Globally first line therapy
 eradication treatment recommended for H. pylori, combines a proton-pump inhibitor (PPI) with
 two antibiotics (amoxicillin and clarithromycin or metronidazole). However, today
 effectiveness of this therapy is less than 75% "percentage", due mainly to H. pylori
 antibiotic resistance. Other factors involved in therapeutic failure are treatment adherence
 and host genetic factors that may affect medication pharmacokinetics, specifically PPI
 pharmacokinetics PPI play an essential role in eradication therapies, it suppresses deeply
 acid secretion, thus increasing hydrogen ion potential (pH) above 6.0. This allows H. pylori
 to replicate more actively than when stomach pH is less than 6.0, therefore improves
 antibiotic activity promoting greater antimicrobial agent stability and antibiotic
 concentration in stomach. PPIs action depends on its metabolism by cytochrome P450 (CYP)
 enzymes.Recent studies have showed that CYP2C19 genotype can affect PPIs ability to suppress
 acid secretion in the stomach, as has been observed for omeprazole. Omeprazole is metabolized
 by CYP2C19 (90%"percentage") and by CYP3A4 (10%"percentage"). CYP2C19 gene has 21
 polymorphisms, three of which play an important role in PPIs metabolism. Thus, depending on
 polymorphism they are designated as early metabolizers (EM) ("asterisk"*1/"asterisk"*1),
 intermediate metabolizers (IM) ("asterisk"*1/"asterisk"*X) and poor metabolizers (PM)
 ("asterisk"*X/"asterisk"*X). Where "asterisk"*1 allele is the wild type allele ("Wild type")
 and "asterisk"*X correspond to mutated allele. Patients with genotype CYP2C19
 "asterisk"*1/"asterisk"*2 o"asterisk" *1/"asterisk"*3 are IM and those with genotype CYP2C19
 "asterisk"*2/"asterisk"*2 or CYP2C19 "asterisk"*3/"asterisk"*3 are PM. The presence of
 CYP2C19 "asterisk"*2 and CYP2C19 "asterisk"*3 polymorphisms predict the individual
 metabolizer phenotype. In 2006 the CYP2C19"asterisk"*17 allele was found, whose function is
 clinically important because heterozygotes, with CYP2C19"asterisk"*1/"asterisk"*17 forms are
 simply considered ultra-fast metabolizers by other authors. The clinical implication of this
 latter polymorphism relies on the following: if PPIs are quickly metabolized, standard PPIs
 dose fails to adequately suppress acid secretion, and H. pylori eradication therapy will be
 less effective if the microorganism is sensitive to antibiotics. A recent work in Colombia to
 eradicate H. pylori, using triple therapy with clarithromycin or levofloxacin combined with
 amoxicillin, reported that although the organism was sensitive to these antibiotics, there
 was variability with the achieved eradication. These results may suggest the possibility that
 additional factors, different from antimicrobial resistance, can influence the treatment
 effectiveness in our population.

 General Methods:

 The aim of present was to determine CYP2C19 genetic polymorphism influence on H.pylori
 eradication. For this purpose a randomized single blinded clinical trial was done. It was
 conducted from 2012 to 2015 in Bogotá, Colombia. The study was performed in accordance with
 Good Clinical Practice guidelines and ethical principles of the Declaration of Helsinki.
 Ethics Committee of the participating institutions approved the study protocol, where all
 participants signed written informed consent.

 After obtaining informed consent, 356 patients were invited to participate in the study. All
 enrolled participants underwent initial endoscopy, carried-out at the upper endoscopy
 service, clinical center from Bogotá, D.C., Colombia to obtain gastric biopsy for histology,
 microbiological and genotypic test. However, due to the strict inclusion and exclusion
 criteria proposed, of the 356 participants, only 133 were included in the study.

 All participants in the study underwent endoscopy of upper digestive tract under aseptic
 conditions, with a minimum of six hours of fasting. During the endoscopy five biopsies were
 taken from the antrum and four biopsies from the body for histopathology, microbiology and
 molecular analysis. For histopathological analysis, the biopsy samples were sent to pathology
 in separate properly marked containers. For microbiological and molecular analyses three
 antral and three body of the stomach biopsies were collected and used as follows: one biopsy
 from antrum for rapid urease test, two biopsies (antrum and one from the body of the stomach)
 for H. pylori culture and susceptibility test. Culturing was carried out only when one or
 both additional tests were positive for H. pylori. If the culture was negative, but any other
 tests were positive, microorganism detection was confirmed by molecular biology analysis
 (ureA gene detection H. pylori). It was considered a participant was infected with H. pylori
 when two or more tests were positive. Another biopsy was used for DNA extraction and
 subsequent analysis of CYP2C19 genetic polymorphism.

 Culture was performed from biopsies of participants whose rapid urease test and histology
 were positive, from a biopsy of antrum and body of the stomach on Brucella agar (BD) enriched
 with 7% "percentage" v/v horse blood, Isovitalex (BD) and Helicobacter Pylori Selective
 Supplement (Dent) (Oxoid). Following, the samples were incubated at 37°C with 11%
 "percentage"carbon dioxide (CO2) for 3 to 5 days. Antibiotic susceptibility test from pure
 colonies was carried-out by agar dilution (gold standard method) according to the Clinical
 and Laboratory Standards Institute (CLSI) to determine amoxicillin and clarithromycin minimum
 inhibitory concentration (MIC). In addition, CYP2C19 polymorphism
 "asterisk"*1,"asterisk"*2,"asterisk"*3 and "asterisk"*17 were identified for all 133 patients
 included in this study. For this propose DNA was obtained from gastric biopsies by DNA
 Isolation from Human Samples from commercial Kit (QIAGEN), and then the identification of the
 respective polymorphisms was done.

 CYP2C19 "asterisk"*1,"asterisk"*2,"asterisk"*3 polymorphisms were determined by real-time
 polymerase chain reaction (RT-PCR) using Modular Assays kit for human CYP2C19"asterisk"*2 and
 CYP2C19"asterisk"*3 (Roche), according to manufacturer's instructions. And nested PCR and
 Restriction Fragment Length Polymorphism (RFLP) was done for determination of allele
 "asterisk"*17 according with Baldwin et al. report.

 Due to the study compared two types of therapies, the 133 patients were randomized for
 received the treatments into two groups by a computer program. Group one or conventional
 group and group 2 or personalized group. Both groups received amoxicillin and clarithromycin
 but first group received standard doses of omeprazole and in the second group omeprazole
 doses was adjusting according to the CYP2C19 polymorphism of each participant.

 After performing microbiological and molecular testing, the participants were allocated to a
 treatment regimen according to a randomized crossover sequence, provided by a
 computer-generated randomization list. Participants were contacted by telephone for assigned
 an appointment for treatment delivery according to the assigned group.

 All participants were given detailed information about the adverse effects that could occur
 with the different medications. Verification of possible adverse effects was performed
 telephonically using a validated and pre-coded questionnaire that included the following
 symptoms: diarrhea, metallic taste, nausea, bloated feeling, loss of appetite, vomiting,
 abdominal pain, constipation, and rash. The intensity of each symptom was graded from zero to
 three: 0: absent, 3: severe, using a Likert scale. Finally H. pylori eradication was verified
 8 weeks post treatment by antigen test (Meridian®). For this test, each participants was
 requested a stool sample. The test was carried-out according to manufacturer's indications.

 For the analysis of results, a database of the participants in excel and in spss was
 generated. These databases included general characteristics of each participant, result of
 each of the microbiology tests, molecular biology. Descriptive statistics and analysis of the
 effectiveness of the treatments used were carried out.
Inclusion Criteria:

 -  Subjects with functional dyspepsia or peptic ulcers

 -  Subjects between the ages of 19 and 70 years old

 -  Subjects who were referred for upper endoscopy, and had not received previous H.
 pylori eradication treatment within the last six months.

 -  Subjects who had not received anti-secreting acid, bismuth or antibiotics for other
 diseases 15 days before the endoscopy.

 -  For the study, only patients with sensitive isolates of H.pylori to amoxicillin and
 clarithromycin were included.

 Exclusion Criteria:

 -  Patients with serious comorbidities.

 -  Pregnant women.

 -  Patients allergic to the medications used in this study.