Abstract of medical phd thesis: research on recrudescence and reinfection helicobacter pylori by determination of urecgene in the duodenal ulcer patients

On the other hand, reinfection is defined as a state wherein a patient becomes infected with a new H.pylori strain after the patient went through successful eradication therapy.. Analys

DO NGUYET ANH

RESEARCH ON RECRUDESCENCE AND

REINFECTION HELICOBACTER PYLORI BY DETERMINATION OF UreCGEN E IN THE DUODENAL

ULCER PATIENTS

Speciality: Gastroenterology Code: 62.72.01.43

ABSTRACT OF MEDICAL PHD THESIS

Hà Nội – 2021

THE THESIS WAS DONE IN: 108 INSTITUTE OF CLINICAL MEDICAL AND PHARMACEUTICAL SCIENCES

VIỆN NGHIÊN CỨU KHOA HỌC Y DƯỢC LÂM SÀNG 108

Supervisor:

1 A/Prof PhD Nguyen Thuy Vinh

2.A/Prof PhD Nguyen Thi Hong Hanh

Revie we r:

1

2

3

This thesis w ill be presented at Institute Council at:

108 Institute of Clinical Medica l and Pharmaceutical Sciences

Day Month Year

The thesis can be found at:

1 National Library of Vietnam

2 Library of 108 Institute of Clinical Medical and Pharmaceutical Sciences

INTRODUCTION

Helicobacter pylori (H pylori) is considered to be a major cause of peptic ulcers There are many eradication regimens for H pyloribut there is still a recurrence of H pylori after eradication Many factors influence H pylori reappearance including an important role of H pyloriantibiotic

resistance, factors of socio-economic development and environmental

sanitation conditions.Helicobacter pylori (H.pylori) recurrence can be

classified into two classes: recrudescence and reinfection Recrudescence is

a recurrence of the same infection after antimicrobial treatment, which occurs as first-year relapses On the other hand, reinfection is defined as a

state wherein a patient becomes infected with a new H.pylori strain after

the patient went through successful eradication therapy Distinguishing recrudescence and reinfection helps the gastrointestinal specialty have a strategic direction for treatment That is to change treatment regimen or control risk factors DNAfingerprinting technique is exactly distinguish between recrudescence and reinfection PCR- RFLP and PCR sequencing

determined H.pylori are two comon methods for research and clinical

practice Using above methods for UreC gene analysis is used in many

studies to determine the presence of H.pylori due to its high specificity for H.pylori strains

There have not been many studies on differentiating between

recrudescence and reinfection of H.pyloriby molecular methods inVietnam

Therefore, we conduct this study for two main objectives:

1 Analysis of the rate of resistance to Amoxicillin, Clarithromycin and efficacy of Esomeprazole-Amoxicilin-Clarithromycin (EAC) regimen in Helicobacter pylori positive duodenal ulcer

2 Determinate the recrudescence and reinfection status of Helicobacter pylori after successful eradication treatment, by PCR-RFLP technique and UreC gene sequencing

Thesis layout

The thesis has 127 pages, including: Introduction (2 pages), Chapter 1: Overview (35 pages), Chapter 2: Subjects and methods (20 pages), Chapter 3: Results (32 pages), Chapter 4: Discussion (33 pages), Conclusion (2 pages), Recommendations (1 page) The thesis has 38 tables, 4 Charts, 33 Pictures The thesis has 190 References (19 vietnamese references and 171 english references) and 3 appendices

The ne w main contributions of the thes is

The recurence of H pylori after eradication is quite common in many

countries at different rates.The study identified recrudescence or reinfection

in patients with H pylori re-positive after successful eradication has

important implications for the treatment strategy.This is the first study

using PCR-RFLP technique and genetic sequencing to determine UreC genotype of H pylori strains taken directly from stomach biopsy So that

distinguish recrudescence or reinfection The results of the topic show that the rate of re-infection is higher than recrudescence in the research group

From there, posing the problem of controlling the source of H pylori

infection besides choosing appropriate treatment regimens

CHAPTER 1 OVERVIEW

1.1.Helicobacter pyloriand duode nal ulce r

H.pyloriplays an important role in the pathogenesis of duodenal ulcer

Up to now, there are many combination treatment regimens of 3 drugs,

4 drugs including two antibiotics to eradicate H.pylori, heal ulcers and reduce

ulcers recurrence However, antibiotic resistance, especially Clarithromycin resistance, is one of the reasons that reduce the effectiveness of first line therapy and also make it difficult to treat the next steps

1.2 Recrudescence and Reinfection after H pylori eradication

tre atme nt

After successful eradication treatment, H.pylori still recurence with

recrudescence or reinfection Recrudescence means that a pre-treatment

strain of H.pylori has been eradicated reappeared within 12 months of

follow-up or DNA fingerprinting determines identical strains Reinfection

is the reappearance of H.pylori after 12 months of eradication treatment or

when DNA fingerprinting techniques determine difference strains before and after treatment.In developed countries, recrudescence is common,

H.pylori reinfection tends to be low in developed countries and high in

- Factors related to reinfection, individual susceptibility to

H.pyloriinfection, re-contact with H.pylori is more likely to occur when living in poor sanitary conditions, transmission H.pylori between family members when someone is infected with H.pylori

- Significance of differentiating H.pylori recrudescenceand reinfection

Distinguishing reinfection or recurrence plays an important role in choosing treatment strategy, adjusting second treatment regimen, and controlling risk factors.PCR – RFLP and PCR sequencing can distinguish

subtype H.pylori strains, show the diversity of H.pylori strains From this feature, new research directions can be deployed such as vaccines for H pylori, other drug resistance mechanisms or the interaction mechanism of bacteria with H.pylori infected person

1.3.Methods of distinguishing the genome of Helicobacter pylori and

method of PCR –RFLP, sequencing determine UreC gene

- PCR- RFLP: PCR from gastric biopsy samples to diagnose H.pylori

is high specific method There are several primers designed for gene to

diagnose H.pylori Typing UreC gene is one of the most common gene to

use for diagnosing H.pylori because UreC gene is only in H.pylori but not

in other urease-positive bacteria PCR typing of ureC from H.pylori

isolated from gastric biopsies have specificity from 96% to 100% PCR restric fragment length polymorphism (PCR-RFLP) directly from gastric

biopsy of H.pylori infected patients based on restriction enzyme (HhaI, MboI, Hind III) is a convenient and effective method of detection and pattern of H.pylori without culture

- Gene sequencing: Gene sequencing is to detect the order of these four nucleotides on the DNA molecule Gene sequencing is the most

accurate method of diagnosis for H.pylori

CHAPTER 2 SUBJECTS AND METHODS 2.1 Subjects

303 patients were identified with duodenal ulcer and H

pyloripositive from 5/2012 to 5/2015 at Central Hospital E

2.1.1 Subject selection

- Patient with age ≥16 yeas old:

- Diagnosis of duodenal ulcer when an ulcer image is 0.5 cm or more, has a clear margin

- H pylori infection with criteria: either culture is positive or both

urease test and histopathology are positive

- The patient has not had any treatment for H pylorieradication

- Severe gastrointestinal bleeding, pyloric stenosis, severe renal failure, Child-pugh B or C cirrhosis

- Multiple acute small ulcers

Z

α = 0,05 và Zα/2=1,96 tra từ bảng Z

- Objective2:Total population samplingn = 18

2.2.3.Steps of research process

2.2.3.1 Clinical examination and data collection

Clinical and laboratory data collection following patient’s report at three poit of time: 1st time is the first patient’s meet, 2nd time after treatment 45 days, 3rd time during 1 year (6,9,12 months), after 1year (>12, 18 months, ≥24 months)

- 1st time examination:evaluate clinical symptoms, gastroendoscopy to

assessment of duodenal ulcer lesions,testing to diagnose H pyloriinfection The

patient received the EAC regimen for 10 days

- 2nd time examination:assess pain relief, side effects of medications,

gastroendoscopy to assessment of duodenal ulcer healing, testing to diagnose H pylori infection If patient have H.pylorinegative after eradication treatment will

make an appointment for examination and gastroendoscopy 3rd time after 6 months, 9 months , 12 months, 18 months, ≥24 months

-3rd time examination:Clinical examination, gastroendoscopyto

evaluate ulcer recurrence and H pyloriinfection Patients haveH.pylori

positive (HP+) will be tested PCR - RFLP and genetic sequencing for

H.pylori strain from gastric biopsy At the same time PCR – RFLP and

genetic sequencingforH.pylori strain from gastri biopsy before

treatmentwhich is stored at -200C to compare the similarities and

differences ofH pyloristrains infected before and after treatment From there, determine the reccrudescence or reinfection w ith H pylori

2.2.3.2 Gastroendoscopy and diagnostic H pyloritests

-1st Gastroendoscopyevaluate size, location duodenal ulcer Biopsy

of 6 samples at the antrium and body is used for H pyloridiagnostic test

including rapid urease test, pathology (H&E), culture A part of biosy sample store at -200Cfor PCR If bacteria grow, the colonies will be transformed to antibiotic sensitive test with Amoxicillin and Clathromycin

- 2nd gastroendoscopy evaluate ulcer healing Biopsy 4 gastric and

body samples for H pyloridiagnostic test including rapid Urease tesr and

histopathology (H&E)

- 3rd gastroendoscopy: evaluate ulcer recurrence Biopsy 4 gastric

and body samples for H pyloridiagnostic test including rapid Urease tesr

and histopathology (H&E) and 2 samples were stored –1960C for PCR test

if the H pyloritest turned positive again

2.2.3.3.Culture and Antimicrobial susceptibility testing: The culture was carried out in artificial medium on 5% blood agar under microaerobic conditions placed in an incubator of 370C for 2 to 5 days.Antimicrobial susceptibility testing by Amoxicillin and ClarithromycinE test

2.2.3.4.PCR-RFLP UreC geneof H.pylori

- 18 pairs of biopsy samples before and after treatment were

numbered

- Preparation of Genomic DNA for PCR

- Determination of H pyloriinfection by PCR technique: Amplification of UreC gene segment from DNA extracted by primer pair

UreC - F: 5’- TGG GAC TGA TGG CGT GAG GG- 3’

UreC - R: 5’- AAG GGC GTT TTT AGA TTT TT- 3’

- PCR-based Restriction Fragment Length Polymorphism (RFLP)

Analysis: The second PCR product was digested by enzyme HhaI, HindIII and MboI, electrophoresis on gel agarose 1,5%,illuminated under

ultraviolet light and photographed the results

2.2.3.5 UreC gene sequencing of H pyloristrains infected before and

after eradication treatment: DNA extraction from gastric mucosa biopsy tissue samples then electrophoresis of the PCR product on agarose gel.Sequencing by automatic genetic sequencing machine

2.2.4 Research criteria

2.2.4.1 Clinical features

- Age and age group, gender

- Symptoms:epigastric pain, pain when hungry, heartburn, vomiting, nausea, dyspepsia, other symptom

2.2.4.2 Endoscopic features

Size of the ulcer, ulcer location, number of ulcers, healing ulcers

2.2.4.3 Criteriaof urease, histopathology, culture, antibiotic sensitive

- Urease test: posotive, negative

- H.pylori density: Mild, modrate, marked

- H.pylori culture: bacteria grow or not grow

- Evaluate antibiotic sensitive with Amoxicillin and Clarithromycin

by E-test

2.2.4.4 Assess the side effects of the drugs

- Proportion of patients with and without drug s ide effects

-Percentage of drug side effects occurring during treatment

2.2.4.5 Assess recrudescence and reinfection of H.pylori

- Determination of H pyloristrain by PCR with primer pair of gene UreC

820bp

- ReconfirmH.pyloristrain by PCR UreC gene sequencing

- Compare strains before and after eradication treatment by PCR-RFLP + Recrudescence: RFLP type before and after treatment are identical + Reinfection: RFLP type before and after treatment are different

- Evaluate the results of comparing infected strains before and after

eradication treatment by similar UreCgene sequence of H.pylori before and after

treatment

2.2.5 Equipme nt

-Endoscopy system Olympus CV180

- Urease rapid test use Kit UREASEmade in National Institute of Hygiene and Epidemiology

- Histopathology (H&E) follow the process òHistopathology Department – E Hospital

- Culture bacteriain artificial blood jelly 5% environment microaerobic atmosphere.Antibitic sensitive use E-test Amoxicilin and Clarithromycin (BioMerieux)

- PCR – ProflexTM 96- Well PCR System machine

-Electrophoresis set Takara Mupid®-exu

- Gene sequencing machine 3130xl (Applied Biosystems)

- Drugs for regimen therapy:Esomeprazole 20mg (Nexium), Clarithromycin 500mg (Claritek), Amoxicillin 500mg (Hagimox)

2.2.6 Statistical analysis

- By medical statistical methods, usingSPSS 16.0 for Windows

- Result evaluation PCR-RFLP: Image comparison of DNA

electrophoresis tapescủaH pyloristrains infected before and after eradication treatment with DNA maker in order toevaluationof identical and differences

between H pyloristrain

- UreC gene sequences of H pyloristrains were used to access the

Gene Bank(BLAST-Basic Local Alignment Search Tool) comparison with sequences of correctly identified reference strains, comparing similarity

between two UreC gene sequences of strains before and after treatment

2.3 Research e thics

According to regulations of the Ministry of Health and E Hospital

Urease Test and/or MBH Positive

Urease Test and MBH negative

PCR – RFLP Gene sequencing

Do not choose to

study

Nội soi lại sau 6,9,12,

18, ≥24months

Diagnose duodenal ulcer by astroendoscopy

Positive Urease Test

Histopathology Culture, Antimicrob ial susceptibility testing, keep sample for PCR

Treat by EAC for 10 days

Examination and endoscopy again after 45 days to evaluate treatment

results

Ureas e Tes t and/or

Histopathology Pos itive

Ureas e Tes t and Histopathology negative

Do not choose to

study

Endoscopy to evaluate ulcer recurrence

Recrudescence rate of

H.pylori

Reinfection rate of

H.pylori

Figure 2.8 Research diagram

CHAPTER III RESULTS

3.1 Evaluate of Helicobacter pylori positive duodenal ulce r

tre atme nt with Esome prazole -Amoxicilin-Clarithromycin (EAC) regime n

3.1.1.General characteristics of the research sample

- Age, gender: 51,3,8% male, 46,9%% female The average age of patients40,9±14.The most common age group from 21 to 40 years old accounts for 51.8%

- Symptoms of epigastric pain accounted for 84.8%

- Ulcers with size 0.5 - ≤ 1cm are most common (59,7%) Frontal ulcers

are the majority(70,3%)

- The proportion of patients with mild H pyloriinfection is 86,5%

- The success rate of H pyloricultures was 57.8%

- The rate of H.pylori resistance to Clarithromycin before treatment was

40.6%, the rate of resistance to Amoxicillin before treatment was 24.6%

3.1.2 Evaluate treatment outcome of EAC regimen

In 303 patients, 162 patients came back to the examination and endoscopy after 45 days of treatment Therefore, the study evaluated treatment

outcomes in only 162 patients

- 80.9% of patients have epigastric pain symptom reduction

- 17.9% of patients have side effects of the treatment regimen.The rates

of bitter mouth and nausea were 12.3%, insomnia headache was 1.9%,

urticaria 0.6%, diarrhea 3.1%

Board 3.10 The rate of successful eradication of H pylori(n=162)

Res ult Success (HP - ) Failure (HP + ) Total

Comment: 67,9% of successful H pylorieradication (H

pylorinegative after treatment)is not high rate

Board 3.11 Rate of ulcer healing (n=162)

He aling ulce r res ult He aling

Comment: 76,5%ulcer healingis tỷ lệ tương đối cao

Board 3.12 The relationbetween successful H pylorieradication and ulcer healing (n=162)

He aling ulce r res ult

H.pylori e radication

He aling ulce r

No healing ulce r

OR

6,91 (2,91-16,62) <0,01

* Patients with mild H pyloriinfection had a statistically significant difference compared with the rate of patients with moderate and severe H pyloriinfection(p <0.01)

* Patients with mild H pyloriinfection had a statistically significant difference in H pylorieradication rates (86.1%) compared with the rate of patients with moderate and severe H pyloriinfection (p <0.01)

3.1.3 Effects of resistance to Clarithromycin and Amoxicillin on efficacy of EAC regimen

Out of 162 patients who had re-examined and had a second endoscopy to evaluate the treatment results, only 141 pre-treatment patients successfully cultured and had antimicrobial susceptibility testing with Clarithromycin and Amoxicillin.Therefore, this study only evaluated the