Bộ giáo dục và đào tạo

BỘ GIÁO DỤC VÀ ĐÀO TẠO MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENSE VIET NAM MILIRATY MEDICAL UNIVERSITY PHAM HUU QUOC STUDY ON PARACLINICAL CHARACTERISTICS OF HBV, HCV AND GENOTYPES AMONG D[.]

MINISTRY OF EDUCATION

AND TRAINING

MINISTRY OF DEFENSE VIET NAM MILIRATY MEDICAL UNIVERSITY

PHAM HUU QUOC

STUDY ON PARACLINICAL CHARACTERISTICS OF HBV, HCV AND GENOTYPES AMONG DRUG ADDICT USER AT THE DETOXIFICATION CENTERS IN HO CHI

The research was performed at:

Viet Nam Military Medical University

Scientific instructors:

1 Assoc Prof Ph.D Tran Viet Tu

2 Prof PhD Nguyen Tan Binh

Judge 1: Prof PhD Dao Van Long

Judge 2: Assoc Prof Ph.D Bui Vu Huy

Judge 3: Assoc Prof Ph.D Phan Quoc Hoan

The thesis will be defended before the Thesis Assessment Concil atInstitute level

At, date …… month…… year 2022

Be able to search the thesis at:

1 National library

2 Viet Nam Military Medical University library

3 Central Medical Information Library

LIST OF PUBLICATIONS

1 Phạm Hữu Quốc, Trần Việt Tú, Nguyễn Tấn Bỉnh (2017),

Nghiên cứu đặc điểm ALT và nguy cơ viêm gan ở người đang điều trị cai nghiện trong các trung tâm của Thành phố Hồ Chí Minh, Tạp chí Y học Việt Nam, Tập 460, số 1 tháng 11, trang

210 - 214

2 Pham Huu Quoc, Tran Viet Tu, Nguyen Tan Binh (2018),

Study on prevalence of hepatitis B virus/ hepatitis C virus infection in the injecting drug user in rehabilitation centre in

Ho Chi Minh City, Tạp chí Y – Dược học Quân sự, Tập 7, số

43, trang 158 - 164

3 Phạm Hữu Quốc, Lê Văn Duyệt (2019), Nghiên cứu đặc

điểm đột biến kháng thuốc DAA của vi rút viêm gan C ở đối tượng cai nghiện ma túy tại Thành phố Hồ Chí Minh năm

2014, Tạp chí Y học Việt Nam, Tập 481, số 2 tháng 8, trang 8

– 13

Hepatitis B and C are human infectious diseases caused byhepatitis B virus (HBV) and hepatitis C virus (HCV) with manycomplications and serious consequences, if not monitored andtreated promptly can lead to cirrhosis or liver cancer More than

240 million people in the world were infected with HBV andaround 210 million were chronically infected Of which about 1.2million drug users (DUs) were infected with HBV and 10 millionpeople were infected with HCV Many data reported that theprevalence of HBV, HCV infection among people with DUs ismuch higher than that of the community, so this group of DUs is at

a very high risk of infection The proportion of DUs in Vietnam isaround 170,000 DUs, of which about 45,000 DUs are in Ho ChiMinh City (data recorded in 2004) Many studies in Vietnam havenoted that the prevalence of HBV infection among DUs rangesfrom 20-40% and HCV is 31-97.2%

Hepatitis B virus has low genetic variation and is classifiedinto 10 genotypes, distributed in Southeast Asia mainly genotypes

B and C Clinical studies show that the C genotype responds well

to treatment drugs but has a higher risk of mutation and cancer thangenotype B The hepatitis C virus has a very high level of geneticvariation (about 30-35% of the genome sequence), so it is dividedinto seven major genotypes and about 60 sub-genotypes Testing todetermine the genotype of hepatitis B and C viruses plays animportant role in diagnosis, treatment and prognosis

In treatment, the HBV genotype is significant in theprognosis of cancer risk and drug response efficacy, while theHCV genotype is crucial in making treatment regimens Currently,HCV treatment with direct antiviral drugs (DAA) is highlyeffective and easy to use, but the DAA drug resistance associatedwith gene mutations presents a challenge in treatment In Vietnam,there are few studies on infection rates and genotypiccharacteristics of HBV, HCV among drug addicts as well as genemutations associated with DAA drug resistance in HCV treatment

Therefore, we conducted the research "Study on paraclinical

characteristics, HBV and HCV genotypes among drug addict users at the detoxification centers in Ho Chi Minh City".

1 The objectives

1.1 Evaluation of HBV, HCV prevalence, viral load and liver enzyme activity among drug addicts at detoxification centers

in Ho Chi Minh City (2013-2015).

1.2 Identification of HBV, HCV genotypes and gene mutations associated with DAA drug resistance of HCV in drug users at detoxification center in Ho Chi Minh City (2013-2015)

2 New contributions of the thesis

- This study is one of the few studies that performed hepatitis Band C virus infection in drug addicts Especially, this study hasreported significant data of drug addiction characteristics,prevalence, viral load, liver enzyme activity, genotypes and drugresistance mutations of hepatitis virus This data provides formanagers as well as the health sector with a comprehensivepicture of the status of HBV and HCV infection in the subjects ofDUs so that they can offer appropriate prevention and treatmentsolutions

- This is the first study on gene mutations associated with DAAdrug resistance of HCV in Vietnam This is the premise forconducting in-depth and large-scale studies to identify andclarify the characteristics and mechanism of resistance of HCV

to the treatment drugs

- The technique of gene sequencing to identify mutationsassociated with DAA drug resistance is very important in thediagnosis of HCV drug resistance Therefore, it can be applied inpractice to identify drug resistance mutations in patients whohave not been treated or who have failed treatment

- All data of this study are references for the next studies onhepatitis B and C virus infection in particular and infections forDUs

3 The layout of the thesis

The thesis consists of 152 pages, with 4 chapters: Rationale 02pages, Chapter 1 - Introduction: 31 pages, Chapter 2 - Subjects and

methods 17 pages, Chapter 3 Results 26 pages, Chapter 4 Discussion 32 pages, Conclusion and recommendations 03 pages.The thesis has 31 tables, 14 charts, 02 figures, 01 diagram, 138references including 21 Vietnamese documents and 117 Englishdocuments.

-CHAPTER 1 OVERVIEW 1.1 Current circumstances and trends of drug addict

According to The United Nations Office of Drugs and Crime(UNODC) report, the number of new addictive substancesincreased from 126 compounds in late 2009 to 450 compounds in

2014, an increase of nearly four folds In 2011, an estimated 167 to

315 million people with aged 15-64 (equivalent to 3.6 - 3.9% of theadult population) used at least one drug UNODC estimates that in

2011, there were around 11.2 to 22 million people worldwidebetween the ages of 15 and 64 (accounting for 0.24 - 0.48% of thepopulation) use addictive drugs According to the Ministry ofLabor, War Invalids and Social Affairs, by the end of 2005, therewere over 129.000 DUs with records managing, an increase of36.300 people compared to 2000 In particular, Ho Chi Minh City(HCMC) increased by nearly 10.000 people, Hanoi increased by6.300 people Up to now, there are 64/64 provinces, cities and 90%

of districts, 58% of communes and townships have drug users

1.2 Epidemiology of HBV and HCV infection in drug addicts

In 2011, it was estimated that around 1.2 million people in theworld were chronically infected with HBV (with the HBsAg testpositive) and nearly 6.4 million people were positive for HBcAb.Data on the positive rate of HBsAg among DUs have beenrecorded in 59 countries (accounting for about 73% of the number

of DUs worldwide), showing that the proportion of positiveHBsAg in the DUs group correlates with the HBV infection rate inthe population, with the highest rates found in Asia Globally,about 10 million DUs are infected with HCV, with the presence ofanti-HCV antibodies (HCVAb), of which China is about 1.6

million; The United States is about 1.5 million people, Russia isabout 1.3 million people There is very limited data on theprevalence of HBV, HCV infection among DUs in Vietnam.Several studies show that the prevalence of HBV infection (HBsAgpositive) varies from 20% - 40% and HCV is from 31-97.2% in thegroup of DUs

1.3 Genotype characteristics of HBV, HCV

HBV is divided into different genotypes based ondifferences in the nucleotide sequence of the HBV genome Todate, 10 genotypes of HBV have been identified, denoted from A

to J Each HBV genotype (except genotypes E, G and H) can besubdivided into subgenotypes The genotype of HBV wasdetermined when there was > 8% nucleotide difference in thegenome sequence, while the subtype was determined when therewas 4 - 8% nucleotide difference in the genome sequence.Genotypes B and C are common in Asia Identifying genotypes isextremely important in the diagnosis, treatment and prognosis ofthe disease

So far, seven genotypes and more than 60 subgenotypes ofHCV have been identified, in which each genotype has anucleotide difference of 30-35% of the genome sequence and thesubgenotype has a nucleotide difference of 20-25% genomesequence Genotypes 1, 2 and 3 are distributed globally, whilegenotypes 4, 5 and 6 are only distributed in certain geographicalregions In Vietnam, genotypes 1 and 6 are mainly distributed.Apart from the significance of epidemiological surveillance, theidentification of HCV genotype is extremely important to helppredict the level of drug response, the duration of treatment andthereby making it possible for clinicians to personalize thetreatment for each infected person with HCV strains carryingdifferent genotypes

1.4 DAA drug resistance mutations in hepatitis C virus

A number of new direct-acting antivirals (DAA) drugs havebeen introduced, including: simeprevir, sofosbuvir (SOF),paritaprevir, daclatasvir, ledipasvir (LDV), ombitasvir, dasabuvir

(DSV), grazoprevir and elbasvir, inhibitory effects direct virusreplication at target proteins such as NS3/NS4A protease, NS5Bpolymerase and NS5A protein, these drugs can be used incombination or not in combination with Peg-IFN/RBV treatmentfor hepatitis C.

HCV is one of the viruses that has a very high rate ofreplication, but they lack the proof reading ability of the RNA-dependent RNA polymerase This is the reason why HCV cancreate very fast genetic change rate and in a short time (average 1.7

x 10-3 nucleotides/position/1 year) Among these mutations, someoften lead to resistance to the drugs being used to treat HCV dailyfor the people infected with HCV and thereby create a series ofnew virus strains with stronger resistance

The treatment regimen using DAA drugs is only effectivefor wild-type HCV virus but is not really meaningful for HCVstrains carrying resistance mutations, due to under pressure Theselection of these viruses will quickly produce multiple resistance-associated amino acid variants (RAVs) RAVs are usually formedduring treatment, but a proportion of subjects infected with HCVstrains already have mutations of resistance to natural drugs beforetreatment, so conducting test for RAVs before treatment isextremely important to evaluate, predict and suggest the mosteffective treatment regimen for the subject

1.5 Identification of DAA drug resistance mutations

Application of gene sequencing technology has helpedidentify many mutations related to DAA drug resistance of HCV.Many studies have confirmed an association between RAVs onNS3/4a or NS5b genes prior to treatment and treatment failure.These findings underscore the need for baseline screening beforestarting treatment regimens in people infected with a specific HCVgenotype The occurrence of the S282T variant has been associatedwith a decrease in susceptibility to SOF by 2.4 to 19.4 foldscompared to that in all HCV genotypes RAV appears on NS5B atL159F position in genotype 1 associated with virological failureduring short-term treatment with SOF and ribavirin

CHAPTER 2 SUBJECTS AND METHODS 2.1 Subjects

986 DUs were collected at two drug detoxification centers in HoChi Minh City meeting the research criteria from 1/12013 to12/31/2015

2.2 Criteria for selecting subjects

Subjects were selected based on decisions No.5075/QD-BYT(2007) and 3556/QD-BYT (2014)

2.3 Exclusion criteria

- DUs were not in detoxification centers

- HIV infection and other diseases

- Do not agree to participate in the study

2.4 Methods

2.4.1 Study design: prospective, cross-sectional description 2.4.2 Sample size

Calculate the sample size according to the formula

In which: Zα/2 = 1.96 (95% confidence level)

d: Expected error, choose d = 0.05

p: estimated rate of closure of DUs subjects infected with HCV,HBV

According to many studies, p = 0.14 for HBV infection and p =0.54 for HCV infection

Applying the above formula, the minimum sample size for theHBV study was 186 and the HCV was 382 However, to increasethe sample size, we selected 986 subjects to meet the study criteria

2.4.3 Content and procedures

2.4.3.1 Evaluate HBV, HCV infection rate, viral load and liver

enzyme activity

+ Information gathering: Collecting information about DUs such asage, gender, characteristics of drug use, duration of drug use + HBV, HCV infection rates:

- HBsAg test is used to determine HBV infection Performed atthe Modern Medical Center, Ho Chi Minh City.

- Anti Anti-HCV test is used to determine HCV infection.Performed at the Modern Medical Center, Ho Chi Minh City.+ Viral load:

- Quantify HBV-DNA, HCV-RNA by using Cobas 48 TaqMan(Roche) system This test was performed at 175 Hospital HoChi Minh City Quantitative results are expressed at 3 levels:level 1: below the detection threshold (<37.5 copies/ml forHCV and <116 copies/ml for HBV); level 2: quantify the viralload (ranging from 37.5 to 2.75 x 108 copies/ml for HCV andfrom 116 - 9.89 x 108 copies/ml for HBV); level 3: above thedetection threshold (> 2.75 x 108 copies/ml for HCV and from9.89 x 108 copies/ml for HBV)

+ Liver enzyme activity ALT, AST:

- The liver enzymes (ALT, AST) were tested by using AU680system Clinical Chemistry Analyzer (Beckman Coulter) Thistest was performed at the Modern Medical Center, Ho ChiMinh City The liver enzyme activity is normal when ALT ≤

37 IU/L and AST ≤ 40 IU/L at 37oC

2.4.3.2 Identification of HBV, HCV genotypes and mutationsassociated with DAA drug resistance of HCV

+ Genotype of HBV: performed at 175 Hospital Ho Chi Minh City.The S gene segment of HBV was cloned with specific B2/HS4Rprimers and sequenced on 3130 (Applied Biosystem), the HBVgenotype was identified on the datacase:

https://hivdb.stanford.edu/HBV/HBVseq

+ Genotype of HCV: performed at 175 Hospital, Ho Chi MinhCity The Core gene segment of HCV was cloned by specificA5/S7 primers and sequenced on the 3130 system (AppliedBiosystem), the HCV genotype was identified on the database:

https://hcv.lanl.gov/content/index

+ Gene mutations associated with DAA drug resistance of HCV:performed at the Laboratory Department, National Hospital ofTropical Diseases The NS5B gene segment of HCV was cloned by

NSU/NSI primers and sequenced on 3130 system (AppliedBiosystem) NS5B gene mutations of the HCV samples werecompared with wild-type nucleotide sequences of HCV strainswith GenBank accession number: 1A (NC_004102), 1B(EU781827), 3A (D17763), 3B (D49374), 6A (Y12083), 6E(KY608690), 6H (KY608700).

2.5 Enter, manage and process data

Collected data is entered, managed by Excel 10 software, and processed using specialized software STATA 10.0, the references were analized using Endnote X7.

2.6 Ethics

The study was conducted based on the voluntary participation

of drug addicts who are under intensive rehabilitation at detoxification centers in Ho Chi Minh City Information about subjects, HBsAg, Anti - HCV screening tests, viral load, liver enzymes, genotypes and DAA drug resistance mutations of HCV can only be done with the consent of the subjects The information about the subject, the test results are kept strictly confidential, the research is conducted for scientific purposes, not for any other purpose.

CHAPTER 3 RESULTS 3.1 HBV, HCV infection rates, viral load and liver enzyme activity

3.1.1 Characteristics of subjects

Table 3.1 Age distribution of subjects

Age group Subject Proportion (%)

Table 3.2 Addictive substances, duration of

addiction and route of administration

from 10 - <15 years; the number of drug addict duration under 5years and over 15 years is only 14.8% and 5.2% Drug use routewith 58.1% of cases of injecting drug, only 41.9% combined theinjecting and smoking.

3.1.2 Prevalence of HBV and HCV infection among DUs subjects

Diagram 3.1 Test results for HBsAg and Anti-HCV

Using immunological tests to screen for HBV and HCV,there were 120 cases (12.2%) single positive for HBsAg, 550 cases(55.8%) single positive for Anti-HCV and having 44 cases (4.5%)positive for both HBsAg and Anti-HCV Thus, a total of 714/986(72.4%) of the subjects tested positive for HBV and HCV

3.1.3 Characteristics of HBV and HCV viral load

Table 3.3 Viral load of HBV infected subjects

Possitive Negativee

the detection threshold (<116 copies/ml), 2 samples with a load of

116 - 103 copies/ml, 29 samples with a load of 103 - 105 copies/ml,only 5 samples with the loading of over 105 copies / ml 2) in HBVco-infection group with HCV (44 samples), there were 23 samplesbelow the detection threshold, 1 sample with the load of 116 - 103

copies/ml, 14 samples with the load of 102 - 103 copies/ml and 6samples with the load above 105 copies/ml

Table 3.3 Viral load in HCV infected subjects

105 copies/ml, only 25 samples with the volume of over 105

copies/ml 2) in the HCV infected group co-infected with HBV (44samples), there were 26 samples below the detection threshold, 1sample with the load of 37 - 103 copies/ml, 11 samples with theload of 103 - 105 copies/ml and 6 samples with the load above 105