MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH NATIONAL INSTITUTE OF HYGIENE AND EPIDEMIOLOGY NGUYEN THI VAN ANH MOLECULAR EPIDEMIOLOGY OF TUBERCULOSIS IN VIETNAM (2003-2009) Major :Public Health Code : 62.72.03.01 SUMARY OF PhD THESIS IN PUBLIC HEALH HANOI - 2012 The thesis has been done at the National Institute of Hygiene and Epidemiology Scientific supervisors: 1. Assoc. Prof. Dang DucAnh,PhD. 2. Prof. Tran Van Sang, PhD. Referee 1: Assoc. Prof. Doan HuyHau, PhD., Military Medical Institute Referee 2: Assoc. Prof. Bui Duc Duong, PhD., Ministry of Health Referee 3: Assoc. Prof. Nguyen Thi Tuyen, PhD., Hanoi Medical University The thesis will be defended at the InstitutionalScientific Committee held at the National Institute of Hygiene and Epidemiology. At 9 am, September 19, 2012 The thesis can be found at: 1. The National Library 2. The National Institute of Hygiene and Epidemiology 3. ABBREVIATION AFB Acid Fast Bacillus AIDS Arquired Immuno-deficiency Syndrome CAS Central Asian CR Clustering rate DR Direct Repeat EAI East African Indian EMB Ethambutol INH Isoniazid IS Insertion Sequence LAM Latin American and Mediterranean LSP large-Sequence Polymorphisms MIRUs- VNTR Mycobacterial Interspered Repetitive Units - Variable Number of Tandem Repead MPTR Major Polymorphic Tandem Repeat SNP Synonymous Single Nucleotide polymorphisms PAUP Phylogenetic Analysis Using Parsimony PCR Polymerase Chain Reaction PGRS Polymorphic GC-rich Repetitive Sequence RFLP Restriction Fragment Length Polymorphism RMP Rifampicin SM Streptomycin SpolDB4 Spoligotyping database 4 ST Spoligotype VNM Vietnam WHO World Health Organization 1 INTRODUCTION Tuberculosis (TB) is still the second leading cause of death among infectious diseases, causing about 2 millions deaths per year globally. According to the 2010 report from WHO, Vietnam ranked the 12 th in the 22 TB highest burden countries in the world. In the Western Pacific Region, Vietnam ranked the 3 rd after China and the Philippines in the number of prevalence cases as well as the annual new TB cases. Effective standard regimens rapidly reduced TB incidence in developed countries. Therefore,85% of the total number of TB cases in the world is now in developing countries. In fact, due to the increase of populations, political upheavals, migrations, the increase of drug resistant TB and the development ofHIV/AIDS epidemic,the number TB cases at the presence is higher than at any time in the human history. However,it is profoundly disturbing that the means for combating TB are a 100-year-old diagnostic test, a vaccine that was developed 80 years ago, and drugs that have remained fundamentally unchanged for the past 50 years. These facts underlie the call for new tools. Molecular epidemiology of TB is important in this circumstance because it can providescientific fundaments for the development of new tools and the establishment of more appropriate and effective control strategies. The scientific fundaments include how the differences in M. tuberculosispopulation structures in different regions can effect the TB control and prevention measures including TB treatment,development of new vaccines, anti-TB drugs and diagnostic tools;if there are differences betweenM. tuberculosis isolates in children and those in adults; whether these differences can affect vaccine developments; the role of different M. tuberculosis lineages and genotypes in TB epidemiology in different geographic regions; The transmission dynamic of TB in populations; Risk factors for TB transmission, which ones can be prevented. Studies of molecular epidemiology of TB have been carried out in many parts of the world. In Vietnam, there have been a number of studies using molecular techniques for classification and determination of molecular characteristics of M. tuberculosis to investigate TB situation, the distribution of Beijing genotypes and its association with drug resistance. In our study,different aspects of molecular epidemiology of TB in Vietnam wereinvestigated based on the analyses of the causative bacteria and epidemiological data. The most up to date molecular tools and powerful statistics were used in order to obtain the following aims: 2 SPECIFIC OBJECTIVES 1. Characterize the lineage, sub-lineage distribution of M. tuberculosisin some provinces in the Northern plain and of M. tuberculosis isolates from national hospitals, during 2003-2009. 2. Determine the drug resistance levelsof these different M. tuberculosislineages; determine lineage distribution in M. tuberculosis populations of different drug resistance levels,from national hospitals, during 2005-2008. 3. Investigate molecular characteristics of M. tuberculosis isolates from children, during 2005-2009. STUDIED ISSUES - Molecular biodiversity of M. tuberculosis insome provinces in the Northern plain. - Discovery of TB re-infected cases. - Differences in molecular epidemiology of TB at the 3 studied sites in the North, the Centre and the South. - Drug resistance levels of different M. tuberculosis lineages at the 3 studied sites in the North, the Centre and the South. - Lineage and genotypic distribution of M. tuberculosis population of different drug resistance levels. - Association of different drug resistancesin M. tuberculosis isolates in Vietnam. - Characteristics of M. tuberculosis isolates from children, compared to those from adults. NEW CONTRIBUTIONS IN THE THESIS - The description of the lineage/ sub-lineage distribution of M. tuberculosisthat were circulating in the Northern plain of Vietnam and M. tuberculosis isolates from hospitals at national level, during 2003-2009. - The effects of location (urban vs. rural), region (the Northern plain, the North, the Centre and the South) and sampling (hospital vs. population) on the distribution of M. tuberculosis. - The discovery of TB re-infection in TB treatment and control. - The drug resistance levels of different M. tuberculosis lineages in the 3 studied sites in the North, the Centre and the South; The differences in population structuresof M. tuberculosis of different drug resistance levels; The specific association of different drug resistances in M. tuberculosis isolates in Vietnam. - The level of drug resistance and molecular characteristics of M. tuberculosis isolates from Children,the differences between them and those from adults. 3 THESIS STRUCTURE Introduction: 3 pages Chapter 1. Literature review: 58 pages Chapter 2. Materials and methods: 24pages Chapter 3. Results: 39 pages Chapter 4. Discussion: 40 pages Conclusion: 2 pages Proposition: 1 page Chapter 1 - LITERATURE REVIEW 1.1.TB epidemiology TB is caused by Mycobacterium tuberculosis. It is highly aerobic, requires high levels of oxygen and can grow intra-cellular.M. tuberculosis divides every 12-18 hours, which is extremely slow compared to other bacteria. The diseaseis transmittedmainly through respiratory tract,rarely through oral or skin contacts. The main sources of transmission are TB patients with AFB smear positive. Every year, there are about 9 million new TB cases and 2 million of deaths caused by TB in the world, of which 0.4 million are HIV positive. TB presents in all countries but is most prevalent in Africa (30%) and Asia (55%). Eighty percent of the total cases are in the 22 TB highest burdern countries. The number of annual new MDR-TB is about 0.4-0.5 million,the prevalent numberis 2-3 times higher.Up toJanuary2010,XDR-TB cases were reported from 58 countries. Vietnam ranked the 12 th in the 22 TB highest burden countries in the world and ranked the 3 rd after China and the Philippines in the Western Pacific Region. In 2010,per 100.000 populations, there were36 deaths caused by TB, 333 TB cases of all forms and 200 new TB cases. The detection rate was 54%. The rate of HIV positive among TB patients was 3.2% in 2002.In 2007, it was 4.4%. In 2002, the rate of resistance to any drug was 32.5%,the rateof MDR was 3%in new TB patientsand 23.5% in previously treated patients.In 2009, the rate of MDR was 2.7% in new patients and 19% in previously treated patients. 1.2. Population structure of M. tuberculosis 1.2.1. Genetic structure of M. tuberculosis chromosome Repeat elements, single nucleotid polymorphisms, large sequence polimorphisms in M. tuberculosisare important markersfor classification of M. tuberculosis. Insertion sequences (IS)are small fragments of less than 2.5 kb commonly found in M. tuberculosischromosome.Polymorphic GC- 4 rich repetitive sequences (PGRS) are sequences carry a number of tandem repeat 96 bp GC-rich sequences, presentin at leat 26 locations. Direct repeat (DR) region consists of variable number of 36 bprepetitive sequences separated by non-repetitiveunique spacer sequences with length varies from27 to 41 bp.Major polymorphic tandem repeats(MPTR) consist of 10 bp repetitive sequences separated by 5bp spacer sequences. The number of MPTR can be up to 80 copies. Mycobacterial interspersed repetitive units of variable number of tandem repeats (MIRU- VNTR)consist of variable number of 40-100 bptandem repeat units,interspersed in M. tuberculosis chromosome. Single nucleotide polymorphism (SNP)is a variation occurring when a single nucleotide differs between different individuals. Large sequence polymorphism (LSP)is a variation created by insertion, deletion or re- arrangement of a large sequence. 1.2.2. Lineage distribution in M. tuberculosispopulation structures All research on M. tuberculosis population structureshad the same result that the bacteria havelineage distribution in population structures. 1.2.3. M. tuberculosishasa global geographicalstructure Lineage distributions of M. tuberculosis are different in different geographic areas. 1.3. Molecular epidemiology of TB – Applications and achievements 1.3.1. Definition of molecular epidemiology of TB Molecular epidemiology is a field that has emerged from the integration of molecular biology, clinical medicine, statistics and epidemiology. In essence, molecular epidemiology is the use of a multidisciplinary approach for identifying factors that determine the causation, propagation, dissemination, spatial and chronological development of the disease. It focuses on the role of genetic and environmental risk factors, on disease etiology and distribution among host populations at molecular level. 1.3.2. The development history of molecular epidemiology of TB M. tuberculosisgenotyping has been applied since the 90s of the last century. At the beginning it was used for investigations of TB outbreaks and institutional TB transmission. After that applicationswereexpanded to large populations and inter-populations for the determination of recent transmission and risk factors. Molecular epidemiology of TB was then applied for investigations of laboratory cross contaminations, rates of relapses and re-infections among previously treated patients, infectiousness of AFB smear (-) patients,chains of transmission,and transmission of drug resistant or highly virulent strains. 5 It was also used forcontact tracing,studying population structure of M. tuberculosis, the association between population structure changes andenvironmental, climate anddemographic changes as well as applications of control measures. 1.3.3. Applications and achievements in molecular epidemiology of TB 1.3.3.1. ControlTB bymolecular epidemiology of TB:Molecular epidemiology of TB provides information on TB situation, speculatesits future development and determines risk factors for TB transmission. 1.3.3.2.Determination of TB transmission indexes:Discoverchains of transmission by moleculartyping, then determine TB transmission indexes. 1.3.3.3. Investigation of contacts,sources and routes of transmission:Identical or similar genotypes are the clues for identifying cases in transmission chains. This can help investigate contacts, sources and routes of transmission. 1.3.3.4. Investigation of transmission capacity of drug resistant strains: Transmission capacity of drug resistant strains can be determined by the frequency thatthe strains occur in molecular clusters. 1.3.3.5. Tracing particular strains or strains causing outbreaks:Particular strains or strains causing outbreak can be tracted by molecular markers. 1.3.3.6. Investigation of multiple infections:A TB patients can be infected with more than one M. tuberculosis strainsat the same time. This is multiple infection. Multiple infection can be determined by molecular typing. 1.3.3.7. Estimation of TB burdencaused by re-infection: Re-infection is the casethat is re-infected with a new M. tuberculosis strain after being infected with another strain previously. Re-infection can be determined by molecular typing. 1.3.3.8. Investigation of M. tuberculosispopulation structuresand the importanceof different lineages:Molecular typing could identify main M. tuberculosis lineages that are circulating in the world such asBeijing, Haarlem, CAS, EAI, LAM, Manu, S, T, X,and sub-lineages that are specific for countries or regions such as: Manila, Delhi, (VNM) Việt Nam, Northaburi, SOM (Somali), BDG (Bangladesh) etc Different lineages and sub-lineages affect TB situationin each countryorregion differently.Their importance can be investigated by their ablility to spread in the populations which is expressed by their proportions and clustering rates. 1.3.3.9. Development of diagnosis tools:The differences in individual genetic structuresneed to be considered when selecting molecular markers or antigens for the development of diagnosis tools. Sensitivity or specificity 6 can be affected by mutations or molecular diversity of M. tuberculosis population. 1.3.3.10. Development of anti-TB drugs:M. tuberculosis population structures also needto be taken into account when looking for new and potential anti-TB drugs. It is necessary to discover lineages, sub-lineage that may contain SNPs, LSPsthat cause natural resistancesto the drugs in M. tuberculosis. 1.3.3.11. Development of new vaccines:Vaccine efficacy in differentgeographic areas can be different because local M. tuberculosis populations and immunogenicity of the local host populations are not the same. 1.4. Genotyping techniques used forstudies ofmolecular epidemiology of TB 1.4.1. IS6110- RFLPtyping Restriction enzyme PvuII is used to cut M. tuberculosis chromosome at a site onall IS6110. The restriction fragments are then hybridized with probes. Hybridized fragments are visualized by exposedto an optic film. Theresulted RFLP pattern used for classification is specific for each M. tuberculosis strain. RFLP-IS6110 typing has been considered the gold standard for M. tuberculosis classification. However, the technique is complicated, labour intensive and time consuming. It can not be used in quick respond. In additon, it is not efficient for the classification of strains with low copy number of IS6110. 1.4.2. Spoligotyping Spoligotyping is based on the detection of 43 specific spacers in the DR region. The spacers are amplified by PCR using Dra and Drb primers, of which Dra is biotinylated.PCR products are then hybridized on a membrane coated with 43 oligonucleotides that are supplementary with the 43 spacers. The spacers presenting in M. tuberculosis strains hybridized with the oligos on the membrane can be visualized by exposing the membrane with an optic film. Spoligo pattern (spoligotype) is specific for each strainand can be used for classification. Spoligotyping is fast, simple and particularly effective foridentifying Beijing strains. However, its discriminatroty power is lower than IS6110-RFLP. Internationalpoligotypingdatabases and softwares for ananlysis include SpolDB4,SITVIT WEB,SPOTCLUST,SpolTools. 1.4.3. MIRU- VNTRtyping Repetitive sequence of each MIRU-VNTR is amplified and detected by agarose electrophoresis.Based on the size of the PCR product, the repeat at each MIRU-VNTR can be determined. MIRU-VNTR type is presented bya 7 number of 12, 15 or 24digit-format if using 12, 15 or 24 MIRUs-VNTR for typing.Each digit of the MIRU-VNTR type corresponds to the repeat at each MIRU-VNTR.Sometimes, the repeat at one MIRU-VNTR can be equal to or more than 10, leading to 2 digits for one MIRU-VNTR. MIRU-VNTR typing has high discriminatory power. MIRU-VNTR combined with spoligotyping can replace the gold standard IS6110-RFLP typing. Softwares for ananlysis include: MIRU-VNTRplus,eBURST,Bionumerics and PAUP. Chapter 2 - MATERIALS AND METHODS 2.1. Studied subjects.place and time The studied subjects are 5 collections of M. tuberculosis isolates. The first collection includes221 isolates from 8 district TB prevention centres of HaTay andHaNoi provinces and from a general population of Hung Yen province. The second collection includes 300 isolates randomly selected (usingcontinuous patients’ hospitalizedregistration number) from the three national hospitals for TB treatment in the three main regions: the North, the Centre and the South of Vietnam.The third collection includes400 isolates selected by drug resistance criteria from the two national hospitals for TB treatment in the North and the South. The fourth collection includes 19 isolates from children, and the fifth collection includes7 paired isolates collected before and after 6 months of treatment fromthe same patients in Chuong My, Ha Tay. All the studied isolates were collected during 2003- 2009. 2.2. Methodologies 2.2.1. Study design The study is a descriptive study using specialized softwares for the analyses of molecular genotyping results. Genotyping techniques used for this study include spoligotyping and MIRU-VNTR typing. 2.2.2. Sample size The formula for calculating the sample size for the estimation of the proportion of isolates belonging to Beijing lineage(an important lineagein TB epidemiologyin Vietnam). The same sample size was applied for objective 1 and 2 of the study. n: Minimum sample size ε = 10% (Relative error) Z (1-/2) = 1.96 (Confidence coefficient withlevel of confidence of 95%) P = 54% (The proportion of Beijing isolates in Vietnam. Anh D. D.Emerg Infect Dis, 2000) 8 According to the formula, the minimum sample size is 328 for each studied objective. 2.2.4. Studied variables and outcome measures 2.2.4.1. Studied variables - M. tuberculosis lineages and spoligotypes based on spoligotyping. - MIRU-VNTR types based on MIRU-VNTR typing. - Drug susceptibility profiles based on drug susceptibility testing for 4 anti-TB drugs used as first line drugs: RMP,INH, SM and EMB. 2.2.4.2. Outcome measures * To meet the fist objective:“Characterize the lineage, sub-lineage distribution of M. tuberculosis in some provinces in the Northern plain and of M. tuberculosis isolates from national hospitals, during 2003-2009” - Lineage, sub-lineage distribution in M. tuberculosis populations. - The association between the proportion of different lineages and epidemiological factors to identify the factors that associate with the transmission of different M. tuberculosis lineages. - The association between molecular clustering rates and epidemiological factors to identify the risk factors for the transmission of TB. - Molecular clustering rates of different M. tuberculosis lineages, sub- lineages in function of epidemiological factors to investigatetransmission capacity ofdifferent M. tuberculosis lineages, sub-lineages. - Cases of TB re-infection. * To meet the second objective: “Determine the drug resistance levels of these different M. tuberculosis lineages, determine lineage distribution in M. tuberculosis populations of different drug resistance levels, from national hospitals, during 2005-2008.” - The level of drug resistances of different M. tuberculosis lineages in function of epidemiological factors to identify the most resistant lineages and the risk factors for drug resistances. - Lineage distribution in M. tuberculosis populations of different drug resistance levels in function of epidemiological factors to identify the evolution of population structures under drug presence pressure. - The association of different drug resistances with each other in M. tuberculosis population in Vietnam in function of location and M. tuberculosis lineages. * To meet the third objective: “Investigate molecular characteristics of M. tuberculosis isolates from children, during 2005-2009.” - Molecular and drug resistance characteristics in M. tuberculosis isolates from children. - Differences between M. tuberculosis isolates from children and M. tuberculosis isolates from adults. 9 2.2.5. Techniques used in the study 2.2.5.1.Data collection Information onM. tuberculosis isolates was extracted from patients’ clinical records and laboratory test records. 2.2.5.2. Culture of M. tuberculosis isolates M. tuberculosis isolates were cultured from sputum specimens using modifiedPetroffmethod. 2.2.5.3. Drug susceptibility testing Drug susceptibility of M. tuberculosis isolates was determined by Canetti-Grosset proportion test. 2.2.5.4. Spoligotyping Spoligotyping was done following the international standard procedure. 2.2.5.5.MIRU-VNTR typing 12- and 15-MIRU-VNTR typing was done following the international standard procedures. 2.2.6. Data analyses NJ phylogenic tree andminimum-spanning tree were build by MIRU- VNTRplus based on MIRU-VNTR typing. A molecular cluster was defined as a cluster of two or more isolates having identical genotypes by both spoligotyping and MIRU-VNTR typing. Recent transmission can be approximated by the clustering rate (CR). CR = (n c -c)/n where n c is the total number of clustered isolates, c is the total number of clusters and n is the total number of isolates. M. tuberculosis isolates were classified into three groupscorrespond to 3 values of lineage variable: “Beijing”,“EAI4-VNM” and “Others” (i.e. other than Beijing and EAI4-VNM)or “Beijing”, “EAI” and “Others” (i.e. other than Beijing and EAI). Multivariate logistic regression (with logit links) and variance analyses were used to investigate how (i) age, (ii) sex of the patient, (iii) location (urban vs. rural), and (iv) sampling strategy (population-based vs. hospital- based) can affect (i) the proportion of isolates of different genotypes and (ii) the proportion of clustered isolates of different genotypes. Association of different drug resistances in M. tuberculosis was analysedby sorting the isolates based on their drug resistance profiles to 4 first line drugsRMP,INH, SM and EMB. The sorting was conducted usingthe method applied for community ecology (Atmar& Patterson, 1993 and Almeida-Netoet al 2008). The statistics of the resulting pattern is computed and its significativity is calculated by random permutations of 10 the resistances for each individual. One thousand of such permutations proved to be enough to reach stationarity in the statistics distribution, from which we could calculate the p-value of our observed pattern. All the statistical analyses were performed with the R software (R Development Core Team, 2010). 2.3. Study limitation The subjects of the study wereM. tuberculosis isolates. Because the capacity for culture of M. tuberculosis isolates is still limited in Vietnam, the studied isolates mainly came from hospitals. 2.4. Ethics The study was based on the analyses of M. tuberculosis isolates from TB patients.It thus did no harm to the patients. All patients’ information was kept confidential. The study was conducted at highest-level research and specialized institutions in Vietnam and France, by qualified researchers and based on international standardized techniques. Thus, the quality of the study can be assured. Chapter 3 - RESULTS 3.1. Molecular biodiversity of M. tuberculosis isolates circulating in some provinces in Northern plain and of M. tuberculosis isolates from national hospitals, during 2003-2009 3.1.1.Molecular biodiversity of M. tuberculosis isolates from some provinces in Northern plain Table3.2. Lineage/sub-lineage distribution based on spoligotyingof M. tuberculosisisolates from some provinces in Northern plain Hospitals Population Lineage Sub - lineage Urban # (%) Rural # (%) Rural # (%) T otal # (%) Beijing 33 (58 . 9) 30 (28 . 8) 19 (31 . 1) 82 (37 . 1) Beijing - Like 2 ( 3 . 6) 1 (1 . 6) 3 (1 . 4) Beijing 35 (62 . 5) 30 (28 . 8) 20 (32 . 8) 85 (38 . 5) EAI1_SOM 3 (2 . 9) 3 (1 . 4) EAI4_VNM 9 (16 . 1) 32 (30 . 8) 15 (24 . 6) 56 (25 . 3) EAI5 3 (5 . 4) 11 (10 . 6) 12 (19 . 7) 26 (11 . 7) EAI 12 (21 . 4) 46 (44 . 3) 27 (44 . 3) 85 (38 . 5) H H3 2 (3 . 6) 1 (1 . 6) 3 (1 . 4) LAM LAM9 3 (2 . 9) 1 (1 . 6) 4 (1 . 8) MANU1 2 (1 . 9) 1 (1 . 6) 3 (1 . 4) MANU2 2 (1 . 9) 2 (0 . 9) MANU 4 (3 . 8) 1 (1 . 6) 5 (2 . 3) 11 T1 4 (7 . 1) 9 (8 . 6) 1 (1 . 6) 14 (6 . 3) T2 2 (3 . 3) 2 (0 . 9) T2 - T3 1 (1 . 8) 1 (1) 2 (0 . 9) T 5 (8 . 9) 9 (8 . 6) 3 (4 . 9) 17 (8 . 1) X X3 2 (3 . 3) 2 (0 . 9) ZERO 1 (1 . 6) 1 (0 . 5) U 2 (3 . 6) 12 (11 . 5) 5 (8 . 2) 19 (8 . 6) T otal 56 (100) 104 (100) 61 (100) 221 (100) A total of 221 M. tuberculosis isolates were analysed by different typing schemes. The spoligotyping generated 53 spoligo patterns, Ofwhich 38 patterns were found in the SITVIT WEB and 15 patterns, designated as spoligotype nvn1 to nvn15, were not found in the database. The upper clades of the nvn spoligotypes were defined using SPOTCLUST and revised by MIRU- VNTRplus. We found a cluster of 6 isolates in the same district of Chuong My (Ha Tay province) shared the same new spoligo pattern nvn7. There were 8 lineages and 13 sub-lineages circulating in the studied region. Beijing and EAI lineages were the most predominant. Twenty-one clusters and 32 unique isolates were identified by spoligotyping. The most frequent spoligotypes were SIT1 (Beijing) and SIT139 (EAI4-VNM). The 12 MIRU-VNTR typing further divided the 21 spoligo-clusters into 113 MIRU-VNTR profiles including 21 clusters and 92 unique profiles. The clustering rate was 34.4%. The 21 clusters determined by 12 MIRU-VNTR typing were then typed by 15 MIRU-VNTR typing generating 61 MIRU-VNTR profiles including 16 clusters (52 isolates) and 45 unique profiles. The clustering rate reduced to 16.3%. The sizes of the large Beijingand large EAI4-VNM clusters reduced. The isolates in each cluster of all 14 small clusters of 2-3 isolates identified after 15-MIRU-VNTR typing, were consistently found to be from patients living in the same district. The 6 isolates from Chuong My district, sharing spoligotype nvn7 remained in cluster.Their 15-MIRU- VNTR type and was different from the MIRU-VNTR types of all other strains that were typed and named as M_CMY. MIRU-VNTR types of the isolates belonging to the 2 main Beijing and EAI lineages were very different. The two lineages can be differentiated based on MIRU4 (2 repeat for Beijing/ 6 repeat for EAI) or MIRU24 (1 repeat for Beijing/ 2 repeat for EAI) or MIRU26 (4-10 repeat for Beijing/ 2-3 repeat for EAI). The isolates of Beijing and EAI lineages aggregated in two main branches of the minimum-spanning tree created by MIRU- VNTRplus,based on 12 MIRU-VNTR typing. 3.1.2. Effects of sampling and location on molecular diversity of M. tuberculosis isolates from the Northern plain 12 Analyses showed that lineage/ sub-lineage distributions of M. tuberculosis isolatesin hospital samples and population samples were not significantly different. However,clustering rates (CRs) were higher in the hospital isolates (urban: 17.9%, rural: 19.2%) than in the populationisolates (0%) (p<0.001). The CR of Beijing isolates(25.9%) was higher than that of EAI4-VNM isolates (10.7%) andof the isolates other than Beijing and EAI4-VNM(10.0%) (p<0.001). The proportion of Beijing isolateswas significantly higher in urban areas than in rural areas (OR: 3.8 [95%CI: 1.9 -7.6]),withp<0.001. The proportion of Beijing isolates increased when the patients’ age decreased,the proportion of EAI4-VNM isolates increased when the patients’ age increased withp<0.05 (figure 3.5). Figure3.5.Proportion ofBeijing (A), EAI4-VNM (C) andOther (B) isolates in function of patients’ age and location (urban: red, rural: blue). Female patients (mean age: 60.7 [95%CI: 58.7- 62.7]) were older than male ones (mean age: 45.5 [95%CI 43.5- 47.5]),with p<0.0001.Patients from rural areas (mean age: 52.3 [95%CI: 50.3- 54.3])were older than patients from urban areas (42.8 [95%CI: 40.8- 44.8]), withp<0.05. Patients infected with EAI4-VNM isolates (mean age: 55.6 [95% CI: 53.6- 57.6]) were older than patients infected with Beijing isolates (51.1 [95% CI: 49.1- 53.1]),withp<0.05. 3.1.3. Molecular characteristics of paired isolates collected before and after 6 months of treatment from same patients Except the paired CMY-T0-14 and CMY-T6-14having the same spoligotype,all other 6 pairs had different spoligotypes.In 5 out ofthe above 6 pairs, the isolates collected at 6 months after treatment had the same genotype nvn7. 3.1.4. Differences in molecular epidemiology of TB in the 3 studied sites in the North, the Centre and the South There were 6lineages and 13 sub-lineages of M. tuberculosisin the three studiednational hospitals in the North, the Centre and the South. Beijing(55.4%) and EAI(27.5%) lineageswere the two most predominant lineages. The other 4 lineages accounted for 17.1%,including Haarlem 13 (1%), LAM (1.3%), T (6.4%) and Zero (0.3%).The undesignated U strains accounted for 8%. The sub-lineage EAI4-VNM accounted for 14.8% of the total samples. Theproportion of Beijinglineagewas higher in the North (70.4%) and in the South (68%), lower in the Centre (28%).The proportion of EAI lineage and EAI4_VNMsub-lineage higher in the Centre (59% and 27%), lower in the North (7.1% and 6.1%) and in the South (17% and 11%). The differences among regions were significant (p<0.01).The proportion of Beijing isolates increased when the patients’ age decreased (p<0.001).The proportion of EAI and EAI4-VNM isolates increased when the patients’ age increased (p<0.001). 15MIRU-VNTR typingshowed that isolates from the same regionswere more similar than the ones from different regions and tend to aggregate together in the minimum spanning tree (figure 3.8). MIRU-VNTR types of isolates from the 3 regions were relatively different. Isolates of Beijing and EAI lineages aggregated in two main branches of the minimum-spanning tree created by MIRU-VNTRplus based on 15 MIRU-VNTR typing. There were many Ustrains embedded in the EAI branch. Among the 3 most predominant lineages,the repeats at each MIRU- VNTR for Beijing and EAI were less diverse and the frequency for the presence of a certain repeat often accounted for a major proportionof isolates compared to other repeats.For the T lineage,the repeats at each MIRU-VNTR were more diverse and there wereoften 3-4 repeats occurredpredominantly. Figure3.8.Minimum-spaning tree created by MIRU-VNTRplusbased on 15 MIRU-VNTR typing for the isolates from the North, the Centre and 14 the South. Notes:The smal circles: MIRU-VNTR types.The colors of the circles correspond to isolates from the North (blue),the South (red) and the Centre (yellow).The elips: the location in the minimum spaning tree, where the isolates from the same regions aggregated. Thecolors of the elips correspond to regions: North (blue), Centre (brown), South (red). 3.2. Molecular epidemiology of drug resistant TB 3.2.1.The levels of drug resistances of different M. tuberculosis lineages in the North, the Centre and the South In general, M. tuberculosisof all lineages were most resistant to drugs in the South, and most susceptible to drugs in the Centre. Beijing lineage was most resistant and EAI lineage was mot susceptibleexcept in the South In the South,the level of drug resistance of EAI lineage was very high, even higher than Beijing lineage. Multidrug resistance (MDR) isolates from the Centre were found only in Beijing lineage (21.4%). The proportion of MDR in Beijing isolates from the Centre was significantly lower than that from the North (34.8%) and from the South (36.7%),withp<0.0001. 3.2.2. Lineage distributions in M. tuberculosisisolates of different drug resistance levels from the North and the South Figure 3.10.Proportion of Beijing, EAI lineages andEAI4-VNM sub- lineage in function of drug resistance levels and regions.Notes: XDR: extensively drug resistance, MDR: multidrug resistance, SDR: single drug resistance, SST: drug sensitive,North (red) andSouth (blue). Results showed that, the higher the levels of drug resistances, the higher the proportion of Beijing isolates and the lower the proportion of EAI and EAI4-VNM isolates in the populations. The proportions of these different isolates were, however, not significantly different between MDR and XDR populations. Variance analysis showed that lineage distributions inM. tuberculosispopulations of different drug resistance levels were not significantly different between the North and the South (figure 3.10). 15 3.2.3. Genotype distributions of M. tuberculosis isolates of different drug resistance levels Drug supceptible strains are more genotypically diverse compared to drug resistant strains. In the minimum spanning tree,the strains with higher levels of drug resistance are more aggregated in the centre, the strains with lower levels of drug resistance are more scattered and far away from the centre. 3.2.4. The association of different drug resistances with each other in M. tuberculosis populationin Vietnam M.tuberculosisisolates resistant to EMB are also resistant to RMP,INH and SM.Isolates resistant to RMP are also resistant to INH and SM.Isolates resistant to INH are also resistant to SM(figure 3.12). This pattern was consistently found in M. tuberculosisisolates belonging to different lineages and from different regions of Vietnam (p<0.01). Figure 3.12.Distribution of M. tuberculosis isolates belonging to diferent lineages, from different regionsof Vietnam in function of drug resistance profiles. Notes:each line corresponds to one isolate. each collumn coressponds to one drug: drug resistance (red), drug supceptible (grey). 3.3. Characteristics of M. tuberculosis isolates from children 3.3.1. Characteristics of M. tuberculosis isolates from children All 19 M. tuberculosis isolates from children were drug susceptible. The isolates belonged mainly to Beijing lineage (13 isolates, 68.4%) and T lineage (5 isolates, 26.3%). A total of 4 molecular clusters were found in this small number of isolates, including 1 cluster from Hung Yen province,3 clusters from the National hospital for Lung diseases. The clustering rate for the studied isolates from children was 22%. 16 Figure 3.14. UPMAphylogenic tree created by MIRU-VNTRplus forM. tuberculosis isolates from childrenbased on spoligotying and 15 MIRU- VNTRtyping. 3.3.1. Comparison of M. tuberculosis isolates from children to those from adults M. tuberculosis isolates from childrenwere not different to M. tuberculosis isolates from adults. Chapter 4 - DISCUSSION 4.1. Molecular diversity of M. tuberculosisisolates circulating in some provinces in the Northern plain and M. tuberculosis isolates from national hospitals, during 2003-2009 4.1.1.Molecular epidemiology of TB in some provinces in the Northern plain  Beijing lineage and EAI4-VNM sub-lineage predominant in the Northern plain Results showed that there were a number of different M. tuberculosis lineages circulating in the studiedregion. The two most predominant lineages were Beijing (38.5%) and EAI (38.5%). Other lineages accounted only for a small proportion H (1.4%), LAM (1.8%), T (8.1%), X (0.9%) and MANU (2.3%). The result coincided with the lineage distribution of M. tuberculosis in East Asia as showed in theinternational spoligotyping database. Among the sub-lineages of EAI lineage, EAI4-VNM is a special sub- lineage for Vietnam,accounted for 67% of the total isolates belonging to EAI lineageor 25.3% of the total studied isolates.EAI4-VNM isolates were found with a high proportion in Vietnam (50.7% in the Mekong river delta,BuuTN 2009), but found with very small proportions or not found in other countries. This indicated that EAI4-VNM sub-lineage is specific for Vietnam. 17 The proportion of Beijing was higher in urban areas than in rural areas. (62.5% vs. 28.8%,p<0.0001). But this location effect was not observed on other lineages including EAI lineage, EAI4-VNM sub-lineage and the Other group (isolates other than Beijing and EAI4-VNM).  Sampling effect on the rate of recent transmission Molecular clustering rates (the rate of recent transmission) in the isolates of different sampling strategies were significantly different, with p<0,0001. The molecular clustering rate in the hospital isolates was higher (urban: 17.9% and rural: 19.2%) than in the population isolates (0%). The finding suggested that the hospital isolates maybe more virulent than population isolates. This suggestion is supported by the hypothesis of Anderson, 1982, build based on a prediction model that the strains with the highest transmission capacity would also be the ones producing the most severe symptoms. Another possible cause for the higher clustering rate in the hospital samples could be thatthe majority of TB patients only came to hospitals when the disease became much advanced after long delaysand after transmitting the disease toother people. In a previous study, the average time delay for TB diagnosis in TB patients in Vietnam was12 weeks while the recommended time delay should not be more than 3 weeks.  Beijing lineage was emerging in the population and replacingEAI4-VNM sub-lineage The Beijing lineage was found to be associated with younger age.The proportion of Beijing isolates increased when the patients’ age decreased. This finding coincided with the findings of the previous studies in Vietnam including a study conducted in the Mekong river delta.This confirmed that Beijinglineage was emerging in the population in both the North and the South of Vietnam. Beijinglineage was emerging through modern migrations and travels. In the study on the rural areas of Mekong river delta, Buu TN et al., 2009 suggested that Beijing isolates were spread from Ho Chi Minh city to rural areas. They found that the proportion of Beijing isolates in the population living along the main road leading to Ho Chi Minh city was higher than the overall proportion of Beijing isolates in the whole studied areas. In the contrary,the proportion of isolates belonging to EAI4-VNM sub- lineage wasfound decreased in younger patients. This sub-lineage seems to be disappearing in the population and replaced by Beijing lineage. EAI4-VNM isolates were found the most susceptibleto anti-TB drugs, while in the contrary, Beijing isolates were found the most drug resistant, the replacement of EAI4- VNM isolates by Beijing isolates predicts a more serious TB situation in Vietnam in the future.The association between the proportion of Beijing [...]... sub-lineages of M tuberculosis circulating in Vietnam Beijing and EAI lineages were the two most predominant lineages Beijing lineage accounted for 28% to 70% EAI lineage varied largely from 7%to 59% in different regions In the Northern plain, the proportion of Beijing isolates in urban areas (62.5%) higher than in rural areas (28.8%) EAI4-VNM sub-lineage was most predominant 24 25 among EAI sub-lineages... typing was congruent with spoligotyping, especially for the identification of Beijing and EAI lineages Thus,MIRU-VNTR typing can be used for the classification of Beijing and EAI lineages even without the help of spoligotyping The simplification of genotyping procedure would promote routine molecular typing for M tuberculosis strains in low-income countries where Beijing and EAI lineages are predominant... Asia Beijing lineage was originated from Centre Asia It thenspread to North and East Asia Thus, it seems that the spreadsof the two lineages EAI and Beijing are meeting in Vietnam resulting in the replacement of EAI lineage with Beijing lineage.The replacement would have happened firstly to EAI5 sub-lineage then to EAI4-VNM sub-lineage.This explains why EAI4VNM sub-lineagewas still predominant in areas... like Vietnam Results of MIRU-VNTR typing presented very diverse M tuberculosispopulation in Vietnam Thus, the typing method has high discriminatory power and can be suitable for epidemiological investigations CONCLUSION 1 Lineage, sub-lineage distribution of M tuberculosis in some provinces in the Northern plain and M tuberculosis isolates from national hospitals, during 2003-2009 - There were 8 lineages... for the lineages For the T lineage, there were several certain repeats concomitantly predominatedat each MIRU-VNTR The T isolates were often scattered at the end of EAI and Beijing branches in the minimum-spanning tree This distribution indicates that thislineage consisted of strains withdiverse genotypes 4.2 Molecular epidemiology of drug resistant TB in Vietnam  The levels of drug resistance of different... the proportion of Beijing in this region was still relatively low.Therefore, the collection of M tuberculosisstrains in Hue may bethe collection of local strains The proportion of isolates belonging to EAI4_VNM sub-lineage was not higher than the proportion of EAI5 sub-lineage and not as predominant as showed in the North and the South In other the hand, the EAI5 lineage more predominated in this region... different M tuberculosis lineages in the three studied sites in the North, the Centre and the South The high proportion of drug resistance in the South found not only in Beijing lineage (36.7% MDR) but also in other lineages, including EAI4VNM sub-lineage (45.5% MDR) even though this sub-lineage was very sensitive in other regions.This finding suggests that isolates belonging to this sub-lineage developed... in M tuberculosis from children The high proportion ofBeijing isolates in children coincided with the finding that Beijing strains are emerging in Vietnam M tuberculosis from children were still very sensitive to anti-TB drugs All 19 isolates from children were sensitive to the first line anti-TB drugs RMP,INH, SM and EMB The study found that 8 out of the 19 studied isolates from children were in molecular. .. in other Asian countries except Bangladesh, suggested that TB epidemiology in Vietnam was very different from the TB epidemiology in other countries in the region The hypothesis that Beijing lineage was invading and spreading into Vietnam through modern migration and travel was supported by the fact that MIRU-VNTRtypes of the 5 biggest Beijing clusters in the studied samples were also found predominant... in areas where Beijing isolates accounted for a high proportion, while EAI5 sub-lineage predominated only in the areas where the proportion of Beijing isolates was still low  Molecular characteristics of M tuberculosis of the same lineages in the three regions M tuberculosis of the same lineages in the different regions were relatively different, resulting in low molecular clustering rate (4%) and . MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH NATIONAL INSTITUTE OF HYGIENE AND EPIDEMIOLOGY NGUYEN THI VAN ANH MOLECULAR EPIDEMIOLOGY OF TUBERCULOSIS IN VIETNAM (2003-2009) . decreased. This finding coincided with the findings of the previous studies in Vietnam including a study conducted in the Mekong river delta.This confirmed that Beijinglineage was emerging in the population. proportion (68.4%) in M. tuberculosis from children. The high proportion ofBeijing isolates in children coincided with the finding that Beijing strains are emerging in Vietnam. M. tuberculosis from