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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 AND TRAINING DEFENSE VIET NAM MILIRATY MEDICAL UNIVERSITY MINISTRY OF EDUCATION PHAM HUU QUOC STUDY ON PARACLINICAL CHARACTERISTICS OF HBV, HCV AND GENOTYPES AMONG DRUG ADDICT USER AT THE DETOXIFICATION CENTERS IN HO CHI MINH CITY Specilize: Internal medicine Indentification number: 9720107 SUMMARY OF MEDICAL THESIS HA NOI – 2022 The research was performed at: Viet Nam Military Medical University Scientific instructors: Assoc Prof Ph.D Tran Viet Tu 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 at Institute level At, date …… month…… year 2022 Be able to search the thesis at: National library Viet Nam Military Medical University library Central Medical Information Library LIST OF PUBLICATIONS Phạm Hữu Quốc, Trần Việt Tú, Nguyễn Tấn Bỉnh (2017), Nghiên cứu đặc điểm ALT nguy viêm gan người điều trị cai nghiện trung tâm Thành phố Hồ Chí Minh, Tạp chí Y học Việt Nam, Tập 460, số tháng 11, trang 210 - 214 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 Phạm Hữu Quốc, Lê Văn Duyệt (2019), Nghiên cứu đặc điểm đột biến kháng thuốc DAA vi rút viêm gan C đối tượng cai nghiện ma túy Thành phố Hồ Chí Minh năm 2014, Tạp chí Y học Việt Nam, Tập 481, số tháng 8, trang – 13 RATIONALE Hepatitis B and C are human infectious diseases caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) with many complications and serious consequences, if not monitored and treated promptly can lead to cirrhosis or liver cancer More than 240 million people in the world were infected with HBV and around 210 million were chronically infected Of which about 1.2 million drug users (DUs) were infected with HBV and 10 million people were infected with HCV Many data reported that the prevalence of HBV, HCV infection among people with DUs is much 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 is around 170,000 DUs, of which about 45,000 DUs are in Ho Chi Minh City (data recorded in 2004) Many studies in Vietnam have noted that the prevalence of HBV infection among DUs ranges from 20-40% and HCV is 31-97.2% Hepatitis B virus has low genetic variation and is classified into 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 than genotype B The hepatitis C virus has a very high level of genetic variation (about 30-35% of the genome sequence), so it is divided into seven major genotypes and about 60 sub-genotypes Testing to determine the genotype of hepatitis B and C viruses plays an important role in diagnosis, treatment and prognosis In treatment, the HBV genotype is significant in the prognosis of cancer risk and drug response efficacy, while the HCV genotype is crucial in making treatment regimens Currently, HCV treatment with direct antiviral drugs (DAA) is highly effective and easy to use, but the DAA drug resistance associated with gene mutations presents a challenge in treatment In Vietnam, there are few studies on infection rates and genotypic characteristics of HBV, HCV among drug addicts as well as gene mutations 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" 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) New contributions of the thesis - This study is one of the few studies that performed hepatitis B and C virus infection in drug addicts Especially, this study has reported significant data of drug addiction characteristics, prevalence, viral load, liver enzyme activity, genotypes and drug resistance mutations of hepatitis virus This data provides for managers as well as the health sector with a comprehensive picture of the status of HBV and HCV infection in the subjects of DUs so that they can offer appropriate prevention and treatment solutions - This is the first study on gene mutations associated with DAA drug resistance of HCV in Vietnam This is the premise for conducting in-depth and large-scale studies to identify and clarify the characteristics and mechanism of resistance of HCV to the treatment drugs - The technique of gene sequencing to identify mutations associated with DAA drug resistance is very important in the diagnosis of HCV drug resistance Therefore, it can be applied in practice to identify drug resistance mutations in patients who have not been treated or who have failed treatment - All data of this study are references for the next studies on hepatitis B and C virus infection in particular and infections for DUs The layout of the thesis The thesis consists of 152 pages, with chapters: Rationale 02 pages, Chapter - Introduction: 31 pages, Chapter - Subjects and methods 17 pages, Chapter - Results 26 pages, Chapter Discussion 32 pages, Conclusion and recommendations 03 pages The thesis has 31 tables, 14 charts, 02 figures, 01 diagram, 138 references including 21 Vietnamese documents and 117 English documents CHAPTER 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 substances increased 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 the adult population) used at least one drug UNODC estimates that in 2011, there were around 11.2 to 22 million people worldwide between the ages of 15 and 64 (accounting for 0.24 - 0.48% of the population) use addictive drugs According to the Ministry of Labor, War Invalids and Social Affairs, by the end of 2005, there were over 129.000 DUs with records managing, an increase of 36.300 people compared to 2000 In particular, Ho Chi Minh City (HCMC) increased by nearly 10.000 people, Hanoi increased by 6.300 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 the world were chronically infected with HBV (with the HBsAg test positive) and nearly 6.4 million people were positive for HBcAb Data on the positive rate of HBsAg among DUs have been recorded in 59 countries (accounting for about 73% of the number of DUs worldwide), showing that the proportion of positive HBsAg in the DUs group correlates with the HBV infection rate in the population, with the highest rates found in Asia Globally, about 10 million DUs are infected with HCV, with the presence of anti-HCV antibodies (HCVAb), of which China is about 1.6 million; The United States is about 1.5 million people, Russia is about 1.3 million people There is very limited data on the prevalence of HBV, HCV infection among DUs in Vietnam Several studies show that the prevalence of HBV infection (HBsAg positive) varies from 20% - 40% and HCV is from 31-97.2% in the group of DUs 1.3 Genotype characteristics of HBV, HCV HBV is divided into different genotypes based on differences in the nucleotide sequence of the HBV genome To date, 10 genotypes of HBV have been identified, denoted from A to J Each HBV genotype (except genotypes E, G and H) can be subdivided into subgenotypes The genotype of HBV was determined when there was > 8% nucleotide difference in the genome sequence, while the subtype was determined when there was - 8% nucleotide difference in the genome sequence Genotypes B and C are common in Asia Identifying genotypes is extremely important in the diagnosis, treatment and prognosis of the disease So far, seven genotypes and more than 60 subgenotypes of HCV have been identified, in which each genotype has a nucleotide difference of 30-35% of the genome sequence and the subgenotype has a nucleotide difference of 20-25% genome sequence Genotypes 1, and are distributed globally, while genotypes 4, and are only distributed in certain geographical regions In Vietnam, genotypes and are mainly distributed Apart from the significance of epidemiological surveillance, the identification of HCV genotype is extremely important to help predict the level of drug response, the duration of treatment and thereby making it possible for clinicians to personalize the treatment for each infected person with HCV strains carrying different genotypes 1.4 DAA drug resistance mutations in hepatitis C virus A number of new direct-acting antivirals (DAA) drugs have been introduced, including: simeprevir, sofosbuvir (SOF), paritaprevir, daclatasvir, ledipasvir (LDV), ombitasvir, dasabuvir (DSV), grazoprevir and elbasvir, inhibitory effects direct virus replication at target proteins such as NS3/NS4A protease, NS5B polymerase and NS5A protein, these drugs can be used in combination or not in combination with Peg-IFN/RBV treatment for hepatitis C HCV is one of the viruses that has a very high rate of replication, but they lack the proof reading ability of the RNAdependent RNA polymerase This is the reason why HCV can create very fast genetic change rate and in a short time (average 1.7 x 10-3 nucleotides/position/1 year) Among these mutations, some often lead to resistance to the drugs being used to treat HCV daily for the people infected with HCV and thereby create a series of new virus strains with stronger resistance The treatment regimen using DAA drugs is only effective for wild-type HCV virus but is not really meaningful for HCV strains carrying resistance mutations, due to under pressure The selection of these viruses will quickly produce multiple resistanceassociated amino acid variants (RAVs) RAVs are usually formed during treatment, but a proportion of subjects infected with HCV strains already have mutations of resistance to natural drugs before treatment, so conducting test for RAVs before treatment is extremely important to evaluate, predict and suggest the most effective treatment regimen for the subject 1.5 Identification of DAA drug resistance mutations Application of gene sequencing technology has helped identify many mutations related to DAA drug resistance of HCV Many studies have confirmed an association between RAVs on NS3/4a or NS5b genes prior to treatment and treatment failure These findings underscore the need for baseline screening before starting treatment regimens in people infected with a specific HCV genotype The occurrence of the S282T variant has been associated with a decrease in susceptibility to SOF by 2.4 to 19.4 folds compared to that in all HCV genotypes RAV appears on NS5B at L159F position in genotype associated with virological failure during short-term treatment with SOF and ribavirin CHAPTER SUBJECTS AND METHODS 2.1 Subjects 986 DUs were collected at two drug detoxification centers in Ho Chi Minh City meeting the research criteria from 1/12013 to 12/31/2015 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 the HBV study was 186 and the HCV was 382 However, to increase the 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 as age, gender, characteristics of drug use, duration of drug use + HBV, HCV infection rates: - HBsAg test is used to determine HBV infection Performed at the 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 Ho Chi Minh City Quantitative results are expressed at levels: level 1: below the detection threshold (2 7(1,9) 0(0,0) 12(8,5) 2(8,0) ALT < 250(68,5) 14(77,8) 90(63,4) 14(56,0) (ULN) 1-2 95(26,0) 2(11,1) 29(20,4) 6(24,0) 0,002 >2 20(5,5) 2(11,1) 23(16,2) 5(20,0) Note: 2: medium increase For cases of HCV single infection, at the level of liver enzyme increase 1-2 folds, there was no difference between the levels of viral load However, with the level of liver enzyme increase > folds, it was found that the higher the viral load, the more proportional to the increase in liver enzymes (both ALT and AST) The difference is statistically significant with p 116 copies/ml (including 36 single infections and 21 cases of co-infection with HCV), the results of genotyping test were as follows: single HBV infection group with 41.7% (15 samples) were genotype B and 58.3% (21 samples) were genotypes C Whereas in HBV coinfection group with HCV, 44.6% (11 samples) were genotype B and 55.4% (10 samples) are genotypes C Table 3.8 Genotypic characteristics of HCV Genotype HCV HCV+HBV P n % n % 47 25,4 38,9 0,50 1,1 0,0 127 68,6 0,0 1+6 4,9 11 61,1 Total 185 100 18 100 In a total of 203 cases that had a HCV load above the detection threshold (of which 185 were single infection and 18 were co-infected with HBV), the results of genotyping tests (genotype) were as follows: single HCV infection accounting for the majority being genotype (68.6%); followed by genotype (25.4%) Co-infection of genotype + accounting for 4.9% and genotype accounting for 1.1% of total cases We did not found genotypes 2, and Whereas, in HCV co-infection group with HBV, there were only two genotypes: (39.9% - samples) and + (61.1% - 11 samples) Table 3.9 Comparison of liver enzyme activity between single HBV and HCV infection Liver enzyme AST (ULN) ALT (ULN) Single HBV infection Single HCV infection B C 1+6 n(%) n(%) n(%) n(%) n(%) n(%) 2 1(4,8) 1(2,1) 12(9,4) 1(11,1) 2 3(20,0) 1(4,8) 4(8,5) 26(20,5) Note: 2: medium increase Comparing the increase in liver enzymes between HBV and HCV genotypes showed that the number of HCV infections of genotypes and had higher rate of liver enzyme increase compared with HBV genotypes B and C both in ALT and AST group, especially at the level > folds 3.2.2 The mutations associated with DAA drug resistance of HCV Table 3.10 The mutations associated with DAA drug resistance of HCV Vị trí ĐB WT 1 15 17 18 19 20 21 23 24 26 29 30 32 37 38 39 46 47 27 34 WT 42 WT 6 237 273 282 309 310 316 320 321 326 329 330 333 E G G G G G E D - N Y Y Y Y Q S N R S K S S S - Q R R R R R W W W K R R R S R Q - D G G G K N D N C L T A A C C - L P P P A P P V V L L - V L L A I I V V - S G G G G G G L L L L P P T D E S - S T T T F R S F - Q C C C R R R D Q - A T T T S S E D A - Vị trí ĐB 10 16 22 25 28 36 43 48 11 49 45 50 237 273 282 309 310 316 320 321 326 329 330 333 G - S S S S S S N N - R - R - - H F - G - T K A V P F - R R - A A - Randomly selected 50 HCV samples to identify DAA drug resistance mutations in the NS5B gene Among 27 samples of HCV genotype 1, there were 23 samples carrying mutations on the NS5B gene, the highest mutation rate appeared at amino acid 326 with ~ 60.9% of HCV samples had mutations, followed by at 309 (57%), 320 (35%), 330 (26%), respectively The proportion mutations of HCV samples ranged from 7-21% For HCV genotype samples, only one single sample had mutations in two positions, 273 and 333 Among 21 HCV sample with genotype 6, there were 16 mutant samples, the highest mutation was at 273 with 68,8% In other locations, the proportion of HCV samples with mutations ranged from 6.2-25% CHAPTER DISCUSSION 4.1 HBV, HCV infection proportion, viral load and liver enzyme activity 4.1.1 Prevalence of HBV and HCV infection Prevalence of HBV infection Among 986 DUs subjects with 120 people (12.2%) who tested positive for HBsAg, the proportion of hepatitis B infection was higher than average compared to hepatitis B infection in the community but quite similar to other studies on DUs subjects In the study of Hahné et al (2013) in European countries, the proportion of positive HBsAg in the community in general ranged from 0.1% to 5.6%, this rate ranged from 0.5% - 21.3% in the DUs group Other studies in European countries also noted that the rate of positive HBsAg in the DUs group is much higher than the infection rate in other groups A study by Saraswati L R et al (2015) surveying 2292 DUs men in India found that the prevalence of HBV was 9.7% The high prevalence of HBV infection among DUs shows that the possibility of HBV transmission via DUs is very worrying despite the availability of an effective HBV vaccine and a well-established hepatitis B vaccination program in the community Vaccination against hepatitis B vaccine for high-risk people and HIV-infected people who have not been vaccinated against hepatitis B vaccine is the first measure in managing HIVinfected people to prevent HBV infection in Western countries today, but has not yet been fully tracked and evaluated in Vietnam Prevalence of HCV infection Many studies performed in Vietnam and other countries show that the prevalence of chronic HCV infection in DUs appears to be more common than that of HIV and HBV In this study, 986 DUs subjects who were detoxified in centers in Ho Chi Minh City showed that the rate of anti-HCV positive in the DUs group was 55.8% A number of previous studies in Vietnam also recorded a high prevalence of HCV infection among DUs, such as Nguyen Tien Hoa et al (2012) showed that the positive anti-HCV rate among DUs was 60.0% in 2008, 57.3% in 2009 and 69.3% in 2010, respectively Study by Vu Minh Quan et al (2009) conducted in Bac Ninh province showed that the prevalence of HCV infection among DUs was 74.1% Study by Sereno L et al (2012) HCV infection rate among drug addicts in Vietnam ranged from 31 - 97.2% Study by Nadol P et al (2015) on 3010 DUs in Vietnam recorded 53.8% of people with HCV Thus, the risk of HCV infection in the DUs group is very large, so the prevention of HCV infection for Dus is a major challenge for the health sector as well as the whole society because there has been no preventive