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 Ma
Trang 1MINISTRY 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
Trang 2ABBREVIATION
AFB Acid Fast Bacillus
AIDS Arquired Immuno-deficiency Syndrome
CAS Central Asian
CR Clustering rate
EAI East African Indian
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
SpolDB4 Spoligotyping database 4
WHO World Health Organization
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 12th in the 22
TB highest burden countries in the world In the Western Pacific Region, Vietnam ranked the 3rd 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:
Trang 3SPECIFIC 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
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 12th in the 22 TB highest burden countries in the world and ranked the 3rd 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
Trang 4GC-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
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
Trang 5can 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
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)
Trang 6According 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
- 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
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 = (nc-c)/n where nc 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
Trang 7the 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
Lineage Sub-lineage Urban
# (%)
Rural
# (%)
Rural
# (%)
Total
# (%) 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)
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)
MANU 4 (3.8) 1 (1.6) 5 (2.3)
T1 4 (7.1) 9 (8.6) 1 (1.6) 14 (6.3)
T 5 (8.9) 9 (8.6) 3 (4.9) 17 (8.1)
Total 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-15-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
Trang 8Analyses 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
(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
Trang 9the 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)
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%
Trang 10Figure 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 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
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-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