genetic diversity of multidrug resistant mycobacterium ......instructions for use title genetic...
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Instructions for use
Title Genetic diversity of multidrug resistant Mycobacterium tuberculosis Central Asian Strain isolates in Nepal
Author(s) SHAH, YOGENDRA
Citation 北海道大学. 博士(獣医学) 甲第12849号
Issue Date 2017-09-25
DOI 10.14943/doctoral.k12849
Doc URL http://hdl.handle.net/2115/71081
Type theses (doctoral)
File Information YOGENDRA_SHAH.pdf
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
1
Genetic diversity of multidrug resistant Mycobacterium tuberculosis Central
Asian Strain isolates in Nepal
(ネパールで分離された多剤耐性中央アジア型結核菌株の遺伝的多様性)
Yogendra Shah
2
CONTENTS
ABBREVIATIONS…………………………………………………………………………4
PREFACE…………………………………………………………………………………...6
CHAPTER I
High diversity of multidrug resistant Mycobacterium tuberculosis Central Asian Strain
isolates in Nepal
Introduction…………………………………………………………………………..……….9
Materials and Methods………………………………………………………………………10
-Sample collection and drug susceptibility test
-DNA extraction
-Spoligotyping
-MIRU-VNTR typing
-Sequencing of drug resistance associating genes
-Data management and analysis
-Ethics statement
Results………………………………………………………………………………………..12
-Demographic information
-Spoligotype
-MIRU-VNTR
-Sequencing analysis of drug resistance-associating genes
Discussion…………………………………………………………………………………….14
Summary……………………………………………………………………………...............27
3
CHAPTER II
Characterization of genetic diversity of multidrug resistant Mycobacterium tuberculosis
Central Asian Strain isolates from Nepal and comparison with neighboring countries
Introduction………………………………………………………………………………….28
Materials and Methods………………………………………………………………………29
-Sample collection and drug susceptibility test
-DNA extraction
-Spoligotyping and MIRU-VNTR typing
-Data management and analysis
-Minimum spanning tree (MST)
Results………………………………………..……………………………………………….31
-MIRU-VNTR analysis
-MST analysis
Discussion…………………………………………………………………………………….33
Summary…………………………………………………………………………………. .…43
CONCLUSION………………………………………………………………………………44
ACKNOWLEDGEMENTS…………………………………………………………………46
REFRERENCES…………………………………………………………………………….49
4
ABBREVIATIONS
TB Tuberculosis
MTB Mycobacterium tuberculosis
CAS Central Asian strain
MDR-TB Multi-drug resistant tuberculosis
XDR Extensively drug resistant
DOTS Directly observed short course therapy
WHO World Health Organization
GENETUP German Nepal tuberculosis project
NTP National tuberculosis program
NHRC Nepal Health Research Council
HIV Human immunodeficiency virus
DST Drug susceptibility test
LJ Löwenstein-Jensen
INH Isoniazid
RIF Rifampicin
STR Streptomycin
EMB Ethambutol
RR Rifampicin resistant
rpoB Gene encoding RNA polymerase β-subunit
RRDR Rifampicin resistance determining region
katG Gene encoding catalase-peroxidase
inhA Gene encoding enoyl-acyl carrier protein reductase
WT Wild type
SIT Spoligo-international type
MIRU Mycobacterial interspersed repetitive units
VNTR Variable number of tandem repeats
5
ETR Exact tandem repeats
QUB Queens University Belfast
RFLP Restriction fragment length polymorphism
SNP Single nucleotide polymorphism
LSP Large sequence polymorphism
DR Direct repeat
PCR Polymerase chain reaction
HGDI Hunter Gaston discriminatory index
MST Minimum spanning tree
SPSS Statistical package for the social science
UPGMA Unweighted pair group method with
arithmetic mean
6
PREFACE
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB),
which remains a major public health problem globally as one of the leading causes of mortality
and morbidity in developing countries. Although TB is a preventable and curable disease with
availability of effective anti-tuberculosis drugs for last five decades, the World Health
Organization (WHO) estimates that, in 2015 alone, 10.4 million new cases occurred around the
world, and of these, 1.4 million resulted in death (53). The majority of deaths were reported in
developing countries, with more than half occurring in Asia (58%). TB usually affects lungs
(pulmonary TB), although other organs (extrapulmonary TB) are involved in 15-30% of the
cases (53). The disease is spread when people who are sick with pulmonary TB expel the
bacteria into the air during coughing, sneezing and speaking (8). Overall, a relatively small
proportion (5-15%) of estimated 2-3 billion people infected with MTB will develop active TB
disease. However, the chance of developing TB disease is much higher among people infected
with human immunodeficiency virus (HIV) (53, 55).
Nepal is a landlocked country in South Asia, surrounded by two high TB burden countries,
north by China and to east, south and west by India sharing an open border with India. These
two neighboring countries together account for one third of the world’s TB cases (56). Nepal
experiences every year a large number of TB cases. In 2015, Nepal reported a TB mortality rate
of 21%, and estimates of TB prevalence and incidence were 215 and 156, respectively, per
100,000 inhabitants (53). Despite a TB control program run by the government, the number of
TB cases in Nepal has not decreased in the last decade. The reason behind this phenomenon
remains unknown; thus, comprehensive studies on MTB transmission in Nepal are needed.
The emergence of multidrug resistance (MDR) in TB, resistant to isoniazid (INH) and
rifampicin (RIF), poses a serious threat to the success of TB control programs. In 2015, WHO
estimated that 480,000 new cases of MDR-TB occurred globally. About 45% of the global
MDR-TB cases were from India, China and Russian federation countries (53). The latest (2014)
national drug resistance survey in Nepal revealed that 2.2% in new TB cases and 15.4% re-
treated cases were MDR (33). Studies on drug resistance-associated genes from different
countries have shown that resistance to RIF is due to a point mutation found in the region called
7
RIF resistance-determining region (RRDR) in the β-subunit of RNA polymerase gene (rpoB)
in more than 90% of the cases. RIF resistance can be used as a surrogate marker for MDR-TB
detection. Apparently more than 95% isolates that are RIF resistant are also resistant to INH (5,
19, 37, 39).
Genotyping of MTB isolates has been proven to be a powerful tool for investigating
suspected outbreaks, source of transmission, transmission chain and circulating strains (6, 14).
Spoligotyping (28), mycobacterial interspersed repetitive units-variable number of tandem
repeats (MIRU-VNTR) analysis (46) and IS6110 restriction fragment length polymorphism
(RFLP) typing (7) are commonly used techniques for genotyping. However, IS6110 RFLP
typing is a time-consuming technique and comparison of its results between laboratories is
difficult (48). As a result, spoligotyping and MIRU-VNTR have been used more often in recent
molecular epidemiological studies. Both of these genotyping methods are reliable,
discriminative and technically feasible for comparisons between laboratories (11, 13, 31).
Spoligotyping is based on the detection of 43 unique spacers situated among the direct repeats
at an exact locus of the MTB genome identified as the direct repeat (DR) locus (13).
Mycobacterial interspersed repetitive units (MIRUs) is DNA elements composed of tandemly
repeated sequences and distributed in the bacterial genome (4). The MIRU-VNTR genotyping
method depends on the identification of the difference in number of tandem repeats at several
loci as the repeat number varies among strains. The discriminatory power of MIRU-VNTR
analysis are depends on the number of MIRU loci assessed; 12 loci, 15 loci and 24 loci (10).
The combination of spoligotyping and MIRU-VNTR analysis have been shown to have similar
discriminatory power to IS6110 RFLP.
MTB complex associated with human infections consists of seven lineages based on
specific genetic markers and geographical areas: Lineage 1 (Indo-Oceanic lineage), Lineage 2
(East-Asian Lineage, includes Beijing family), Lineage 3 (East African Indian, includes
Delhi/CAS family), Lineage 4 (Euro-American lineage), Lineage 5 and 6 (M. africanum West
lineages 1 and 2) and Lineage 7 (Ethiopia) (11, 12, 20, 22). Lineage 3, Central Asian strain
(CAS) family has been reported as one of the dominant genotype of MTB in South Asian
countries. Beijing family belonging to Lineage 2 is well known to be associating with drug
8
resistance development, however, CAS family is also suggested to be associating to the
increasing number of MDR TB in South Asian countries (25, 44, 57). In Nepal, there has been
no report about the epidemiology and molecular analysis on MDR CAS MTB, although this
lineage is the majority among TB patients in this country. Thus, I decided to perform the
characterization of the MDR MTB isolates belonging to CAS family in Nepal.
The present thesis consists of two chapters; in chapter I, I have investigated the genetic
characteristics of MDR-MTB CAS family isolates in Nepalese patients. I analyzed 145 MDR
CAS family isolates from Nepal by spoligotyping, 24 loci MIRU-VNTR analysis and drug
resistance-associating gene sequencing. In Chapter II, I have further performed genetic
characterization of MDR CAS family isolates and elucidated evolutionary relationships of
predominant MDR CAS family, CAS_Delhi (SIT26), isolates in Nepal and neighboring
countries (India and Pakistan) by using molecular genotyping tools.
9
Chapter I
High diversity of multidrug resistant Mycobacterium tuberculosis Central Asian
strain isolates in Nepal
Introduction
Previous studies on the genotype of MTB isolates in Nepalese patients reported the Lineage
3 Central Asian Strain (CAS) family as the most dominant in Nepal (29). Other studies also
reported the CAS family as dominant (1, 24, 41, 43) and as contributors to multi-drug resistance
(MDR) in TB in South Asian countries (57). For instance, in India, Stavrum et al. (2009) (44)
associated the CAS family with multi-drug resistant TB (MDR-TB), and in Pakistan it was
linked to the increasing prevalence of MDR-TB and emergence of extensively drug resistant
(XDR) TB (25). It is believed, therefore, that the emergence of MDR-TB in the CAS family
poses a serious threat to the success of TB control programs in the region. Likewise, in Nepal,
MDR-TB is one of the major emerging threats to the success of TB control. For instance, in
2010 a study conducted in Nepal showed a MDR-TB prevalence of 11.7% in re-treated cases
(32), but the latest national anti-TB drug resistance survey conducted between 2011 and 2012
showed that MDR-TB prevalence increased to 15.4% in re-treated cases (33).
The main purpose of the current study is to conduct a genetic analysis of MDR in MTB of
the CAS family and to understand its molecular epidemiological features and transmission
dynamics in Nepalese TB patients.
10
Materials and Methods
Sample collection and drug susceptibility test
A total of 601 MDR-MTB isolates collected from April 2008 to March 2013 by the German-
Nepal Tuberculosis Project (GENETUP) were used, of which 145 MDR-TB CAS family
isolates were purposively selected. Isolates were collected from a decentralized National
Tuberculosis Program (NTP) network of 11 MDR-TB treatment centers and 66 sub-treatment
centers. The location of the treatment centers and the sample numbers collected in each center
are shown in Figure 1. All samples were obtained from different individuals. Epidemiological
features of patients were also collected from hospital medical records. A phenotypic drug
susceptibility test (DST) was carried out using the proportional method on Löwenstein-Jensen
(LJ) medium with standard critical concentrations for isoniazid (INH) (0.2 µg/ml), rifampicin
(RIF) (40 µg/ml), streptomycin (STR) (4 µg/ml) and ethambutol (EMB) (2 µg/ml) (54).
DNA extraction
Mycobacterial colonies on positive LJ cultures were suspended in 300 µl of distilled water
and heated for 20 min at 95 ºC. Heated samples were sonicated in an ultra-sonic water bath
apparatus for 15 min and centrifuged for 5 min at 10,000× g. The bacterial DNA-containing
supernatant was retrieved and used for further molecular analysis.
Spoligotyping
All isolates were analyzed by spoligotyping, as described by Kamerbeek et al. (28). Briefly,
the direct repeat (DR) region was amplified with a pair of primers, and the resulting PCR
products were hybridized to a set of 43 spacer-specific oligonucleotide probes, which were
immobilized on the membrane. Spoligotyping data was analyzed using SITVIT database
(http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE/) to determine spoligo-
international type (SIT) (15).
11
MIRU-VNTR typing
MIRU-VNTR typing was carried out by amplifying 24 loci that included 12 MIRU loci
(MIRU2, MIRU4, MIRU10, MIRU16, MIRU20, MIRU23, MIRU24, MIRU26, MIRU27,
MIRU31, MIRU39 and MIRU40), 4 exact tandem repeats (ETR) loci (ETR-A, ETR-B, ETR-
C and ETR-F), 4 Queens University Belfast (QUB) loci (QUB11a, QUB11b, QUB26 and
QUB4156) and 4 VNTR loci (VNTR424, VNTR1955, VNTR2401 and VNTR3690) as
described by Supply et al. (46).
Sequencing of drug resistance associating genes
Isolates clustered by a combined analysis of spoligotyping and MIRU-VNTR typing were
further analyzed by sequencing of the drug resistance-associated genes, i.e. rifampicin
resistance determining region (RRDR) in rpoB for RIF resistance, katG coding and inhA
promoter regions for INH resistance, as previously described (37).
Data management and analysis
Demographic data including age, sex and treatment history of TB were analyzed with SPSS
(Statistical package for the social science) software version 19.0 and PRISM version 5
(GraphPad Software, Inc., La Jolla, CA, USA). Individual and cumulative Hunter Gaston
discriminatory indices (HGDI) were calculated to determine the discriminatory power of each
MIRU-VNTR locus and overall loci (27). The discriminatory power of each locus was
considered high (HGDI>0.6), moderate (0.3≤ HGDI ≤0.6) and poor (HGDI<0.3) as suggested
by Sola et al. (45). A cluster was defined as two or more isolates sharing the identical
spoligotype and MIRU-VNTR pattern, and clustering rate was calculated using the formula
number of clustered isolates / total number of isolates (23). A phylogenetic tree was constructed
by the unweighted pair group method with arithmetic mean (UPGMA) using an online MIRU-
VNTRplus web-based application [http://www.miru-vntrplus.org] (51).
Ethics statement
This study was ethically approved by the Nepal Health Research Council (NHRC)
12
Results
Demographic information
Age, sex and treatment history information of patients from whom 145 CAS and 456 non-
CAS MDR isolates were obtained are compared and shown in Table 1. Regarding the age group,
a significant difference was found between 0-20 and above 60 years (p < 0.05, test). There
were no other factors significantly associated with the CAS isolate infection among MDR-TB
patients.
Spoligotype
A total of 145 MDR CAS isolates were divided into 25 spoligotype patterns, and
CAS1_Delhi SIT26 was found to be most prevalent (60/145, 41.4%), followed by CAS SIT599
(15/145, 10.3%) and CAS SIT357 (9/145, 6.2%), as listed in Table 2.
MIRU-VNTR
Allelic diversity of each MIRU-VNTR locus and HGDI are shown in Table 3. Three out of
24 loci (QUB26, MIRU10, VNTR424) were found to be highly discriminant, 11 loci (MIRU26,
VNTR3960, MIRU31, MIRU40, ETR-A, QUB11a, QUB4156, VNTR2401, VNTR1955,
MIRU39 and ETR-F) were moderately discriminant and the remaining 10 loci were poorly
discriminant. The number of MIRU-VNTR patterns with the 24 loci was 107 including 21
clusters that consisted of 54 isolates (clustering rate: 37.2 %) (Table 4).
By combining the analysis of spoligotyping and MIRU-VNTR, I found 18 clusters consisting
of 47 isolates with identical pattern in each cluster. The clustering rate was 32.4%, and the
biggest cluster consisted of 6 isolates, followed by three 4-isolate clusters, one 3-isolate cluster,
and finally 13 clusters consisting of 2 isolates (Figure 2, Table 4).
13
Sequence analysis of drug resistance-associating genes
Clustered isolates were further analyzed by sequencing drug resistance-associated genes
(rpoB, katG coding and inhA promoter region) to identify possible MDR-MTB transmission
(Table 5). Among the 47 isolates, all but one had G944C mutation (i.e., Ser315Thr substitution)
in katG, which is a well-known INH resistance-associated mutation. In rpoB analysis, 45
isolates had a mutation or deletion in RRDR, and C1349T (Ser531Leu) was dominant (20/47,
42.6%). Among 18 clusters, 14 clusters had isolates sharing the same rpoB mutation, and half
of them were the major substitution Ser531Leu. Among the remaining clusters, Cluster 1
isolates shared the same 3-codon deletion in which the deletion pattern was identical. In the
biggest cluster, Cluster 13, four isolates shared the same substitution His526Asp and two of
them had a mutation T-8C in the inhA promoter region. In Cluster 6 and Cluster 14, two isolates
each shared a rare substitution, Asp516Phe (GAC→TTC) and Ser531Gln (TCG→CAG),
respectively, both of which require a double mutation in a codon. In Cluster 17, all four isolates
shared a relatively rare substitution, Gln513Leu; moreover, the substitution Leu511Pro shared
in Cluster 11 was also rare. Most of the isolates sharing rare mutations were restricted to the
same geographic locations (Table 5); however, none of those patients had personal contact with
the others sharing the genetically same bacteria. Other demographic features can also be found
in Table 5.
14
Discussion
This study was the first to demonstrate the genetic characteristics of MDR CAS isolates in
Nepal and showed their high diversity. CAS family is reported as being highly prevalent among
TB patients in certain regions of the Indian subcontinent, namely, Pakistan and the northern
half of India (24, 41, 43). Geographically, Nepal is located between two greatly TB-burdened
countries, India and China, which contribute one third of the world’s TB cases. Historically,
Nepal has an open border with India, and both Nepalese and Indian citizens often visit the
bordering area for work, pilgrimage and education. This regular interaction may open an
opportunity for direct transmission of the CAS family MTB between the Indian and Nepalese
populations. In the present study, the ratio of MDR-TB patients infected with the CAS family
was higher in the age group above 60 years than younger groups (Odds ratio 3.25 against 0-20
years old group, p<0.05, Table 1). This association between the CAS family and elderly people
might be related to the historical prevalence of CAS in Nepal and caused by reactivation of
latent TB infection. However, when comparing the patient-age distribution of clustered (n = 47)
and non-clustered (n = 98) isolates, I found that the average age of the clustered-isolate infected
patients was significantly lower than that of non-clustered group (t-test, p<0.05). It may suggest
that younger generations were more likely to be infected with clustered isolates, indicating those
were the outcome of recent transmissions (Figure 3). This phenomenon may be explained by
the higher social activity of younger generations compared with older generations, which likely
contributes to a higher transmission risk.
I found that spoligotyping alone could not identify diversity in the CAS family in Nepal due
to the dominant cluster of CAS1_Delhi SIT26 contributing 60 isolates (Table 2). However, all
145 MDR CAS family isolates were successfully differentiated into 116 patterns including 18
clusters (clustering rate 32.4%) by combining spoligotyping and 24-locus MIRU-VNTR
analyses (Figure 2). I further investigated the optimization of these MIRU-VNTR loci. As
expected, I was able to select an affordable locus set as suggested by previous studies (17, 50).
Based on the cumulative HGDI analysis of 24 loci with clustering rates (Table 4), a set of 15
loci was shown to have the same discrimination power as 24 loci. To get a higher discrimination
power, of the 24 loci reported by Supply et al. (46) I replaced two loci, Mtub29 and Mtub34,
15
with ETR-F and QUB11a. Both of the newly replaced loci were listed among the top 15
discriminant loci that successfully worked for the Nepali MDR CAS cases (Table 4). To save
time and cost, smaller locus numbers of VNTR are better than larger, even though MIRU-
VNTR analysis may be more feasible at local research centers than other typing methods.
In addition, I was able to discriminate clustered MDR isolates by conducting sequencing
analysis of drug resistance-associated genes, katG, rpoB and inhA promoter region. katG 315
substitution is an INH resistance-associated mutation well known for its low fitness cost (49).
However, in highly TB-burdened countries, the majority of INH-resistant MTB isolates have
this mutation, thus it cannot be used as a transmission marker, although, it still can be used as
a genetic marker of INH resistance (5, 37, 39). In contrast, rpoB mutations can be a good marker
for MDR-MTB transmission, as the variation of RFP resistance-associated mutation in RRDR
is much higher than katG in highly TB-burdened countries (5, 37, 39). Having a mutation in
RRDR strongly suggests that the isolate is RFP resistant. RNA polymerase is an essential
enzyme for bacteria, thus the majority of nonsynonymous mutations in RRDR deteriorate and
frequently observed mutations in RFP-resistant MTB are limited. Even such “acceptable”
amino acid substitutions still exert adverse effects on the enzyme function; therefore, RRDR
mutated survivors tend to have additional mutations known as “compensatory mutation” inside
the RRDR or nearby RNA polymerase components (16). In the current study, I found two
double mutations that occurred in one codon. One of them, Asp516Phe (GAC -> TTC), found
in Clusters 6 and 7, may be an example of the aforementioned compensatory mutation, as the
possible parent mutant Asp516Val (GAC -> GTC) was found in both clusters (Table 5). As
Clusters 6 and 7 were different by only one locus with high polymorphism (MIRU10) in the
lineage in MIRU-VNTR (Table 3), and all isolates were obtained from the same geographic
location, the original MDR-MTB bearing Asp516Val may be spread throughout this area. As a
result, some of the descendent bacteria may have acquired an additional mutation in the same
codon, possibly due to the above-mentioned compensatory mutation, which may have been
most suitable for the Asp516Val mutant. Since variety of the compensatory mutation is also
high in RRDR mutant cases (16), the combined analysis of those regions may improve the
detection rate of MDR-MTB transmissions.
16
I found several complete matches of genotypes, geographic locations and drug resistance-
associated mutations between two or more isolates in some clusters, suggesting possible
transmissions of MDR-MTB between individuals (Table 5). In particular, sharing a rare rpoB
mutation in the cluster strongly suggested acquisition of MDR-MTB via person-to-person
contact. I could not identify the exact epidemiological link between those patients, which
suggests that a common transmission source may exist in the geographic area. In some cases,
isolates were obtained from patients living in distant areas. For example, in the case of Cluster
17, four isolates were collected in three different areas, Dharan (East), Nepalgunj (Mid-West)
and Kathmandu (Central), which were as far apart as 200 ~ 600 km. In this cluster, the first case
was an old man in Kathmandu in 2009, and the remaining were young people visiting hospitals
in each area later on. In the case of the patient in the Eastern area of Dharan, it was a new case
of MDR-MTB that showed the same four-drug resistance as the others prior to the TB treatment
(Table 5). As Kathmandu is the capital city, people frequently visit from different areas of Nepal,
and thus, the city could be a site for transmission of infectious diseases (38). This hypothesis,
however, ought to be validated by further whole genome sequencing analysis.
Application of combined analysis of spoligotyping and MIRU-VNTR typing together with
rpoB sequencing is considered an effective approach to conduct molecular epidemiology of
MDR MTB.
17
Figure 1:
Locations of the 11 MDR-TB treatment centers at where samples were collected in Nepal.
A: Mahendranagar, B: Dhangadhi, C: Nepalgunj, D: Bhairahawa, E: Butwal, F: Pokhara, G:
Birgunj, H: Kathmandu, I: Dhanusa, J: Dharan and K: Biratnagar.
Sample numbers collected in each center: (CAS family / Total)
18
19
Figure 2: 24-locus MIRU-VNTR and spoligotyping-based dendrogram of 145 MDR CAS
isolates from Nepal. (A) The dendrogram was generated by UPGMA [www.miru-vntrplus.org].
(B) Strain identification number. (C) SIT (Spoligo-International type) number. (D) Clade
annotated by SITVIT database. (E) MIRU-VNTR results of 24 loci. The order of loci is as
follows, left to right: MIRU2, VNTR424, ETR-C, MIRU4, MIRU40, MIRU10, MIRU16,
VNTR 1955, MIRU20, QUB11b, ETR-A, QUB11a, VNTR 2401, ETR-B, MIRU23, MIRU24,
MIRU26, MIRU27, ETR-F, MIRU31, VNTR 3690, QUB26, QUB4156 and MIRU39. (F)
Spoligotype pattern. Isolates clusters (identical patterns of MIRU-VNTR and spoligotyping)
are enclosed in open boxes.
20
Figure 3: Scattered gram of patient ages for comparison between clustered isolates (n=47) and
non-clustered isolates (n=98) (t-test: P<0.05)
21
Table 1: Demographic and clinical characteristics of patients infected with CAS and non-CAS
genotype isolates.
Number of
CAS family
Number of
non-CAS family
Odds ratio
(95% CI)
p-value
Sex
Male 111 321 1
Female 34 135 0.728 (0.472-1.123) 0.151
Age
0-20 14 73 1
21-40 85 268 1.65 (0.88-3.07) 0.110
41-60 36 99 1.89 (0.95-3.77) 0.066
>60 10 16 3.25 (1.22-8.64) < 0.05a
Treatment status
CAT II failureb 122 393 1
CAT I failurec 18 42 1.38 (0.766-2.48) 0.282
New patients 5 21 0.76 (0.283-2.07) 0.813
a: p-value < 0.05 was considered as significant. Determined by test or Fisher’s exact test.
b: CAT II failure; patients who were either smear-positive relapse, chronic, MDR contact,
relapse and treatment after default
c: CAT I failure; failure in new cases of smear-positive pulmonary TB
22
Table 2: Distribution frequency of diverse CAS family strains
a: Clades were annotated using SITVITWEB database.
b: SIT (Spoligo international types) were assigned by SITVITWEB database and MIRU-VNTR
plus web tool.
c: Closed squares represent positive hybridization (presence of spacer), and open squares
represent no hybridization (absence of spacers).
Cladea SIT
b Spoligotype pattern
c Number of
isolates (%)
1. CAS1_Delhi 26
60 (41.4)
2. CAS 599
15 (10.3)
3. CAS 357
9 (6.20)
4. CAS 142
7 (4.82)
5. CAS 288
7 (4.82)
6. CAS1_Delhi 471
6 (4.13)
7. CAS1_Delhi 1312
4 (2.75)
8. CAS1_Delhi 427
4 (2.75)
9. CAS 22
3 (2.06)
10. CAS1_Delhi 25
3 (2.06)
11. CAS 356
3 (2.06)
12. CAS 486
3 (2.06)
13. CAS1_Delhi 429
2 (1.37)
14. CAS 428
2 (1.37)
15. CAS 485
2 (1.37)
16 CAS 1093
2 (1.37)
17. CAS 1266
2 (1.37)
18. CAS1_Delhi 1343
2 (1.37)
19. CAS1_Delhi 1590
2 (1.37)
20. CAS 203
1 (0.68)
21. CAS 864
1 (0.68)
22. CAS 1151
1 (0.68)
23. CAS1_Delhi 1314
1 (0.68)
24. CAS 1606
1 (0.68)
25. CAS1_Delhi 1883
1 (0.68)
23
Table 3: Allelic diversity of each MIRU-VNTR locus for CAS MDR-MTB isolates of Nepal
(N = 145)
Locus Allele numbera HGDI
c Conclusion
0 1 2 3 4 5 6 7 8 9 10 NDb
QUB26 1 2 4 13 12 89 12 3 0.604 High
MIRU10 2 13 41 83 1 1 3 0.603 High
VNTR424 5 16 88 23 1 1 3 1
0.6 High
MIRU26 1 8 8 20 93 2 12 2 0.56 Moderate
VNTR3690 109 24 11 1 0.465 Moderate
MIRU31 2 1 31 103 5 3 0.448 Moderate
MIRU40 6 23 107 9 0.427 Moderate
ETR-A 1 23 119 2
0.395 Moderate
QUB11a 1 4 2 21 111 3 3 0.394 Moderate
QUB4156 2 2 23
114
4 0.39 Moderate
VNTR2401 119 26
0.351 Moderate
VNTR1955
14 4 116 6 2 3 0.34 Moderate
MIRU39 19 121 3 2 0.322 Moderate
ETR-F 8 124 10 3 0.309 Moderate
MIRU16
23 120 2 0.291 Poor
MIRU20 4 141
0.132 Poor
QUB11b 2 142 1
0.107 Poor
MIRU24 1 144
0.081 Poor
MIRU27 1 140 2 2 0.067 Poor
MIRU4 1 141 1 2 0.054 Poor
MIRU23 1 142 1 1 0.041 Poor
MIRU2 143 1 1 0.027 Poor
ETR-B 145 0 Poor
ETR-C 145 0 Poor
a: Number of tandem repeats
b: ND; Not determined because of PCR failure
c: Hunter Gaston discriminatory index. DI > 0.6: Highly discriminatory; 0.3 ≤ DI ≤ 0.6:
Moderately discriminatory; DI < 0.3 Poorly discriminatory
24
Table 4: Cumulative HGDI with successive addition of each MIRU-VNTR locus (N=145)
VNTR alias Individual
HGDI
No. of
patterns
No. of
clusters
No. of
clustered
isolates
Clustering
rate (%)
No. of
isolates in
each cluster
Cumulative
HGDI
QUB26 0.604 0.604
MIRU10 0.603 18 9 130 89.6 2-52 0.8187
VNTR424 0.6 35 16 121 83.4 2-39 0.8937
MIRU26 0.56 55 20 104 71.7 2-27 0.9453
VNTR3690 0.465 63 20 96 62.2 2-13 0.9674
MIRU31 0.448 70 22 90 62.0 2-11 0.9767
MIRU40 0.427 79 23 82 56.6 2-8 0.9846
ETR-A 0.395 86 25 79 54.4 2-7 0.9874
QUB11a 0.394 90 25 75 51.7 2-6 0.9899
QUB4156 0.39 92 26 73 50.3 2-5 0.9917
VNTR2401 0.351 93 26 71 48.9 2-5 0.9923
VNTR1955 0.34 95 23 62 42.7 2-5 0.9927
MIRU39 0.322 97 22 61 42.0 2-5 0.9927
ETR-F 0.309 97 22 60 41.3 2-5 0.9931
MIRU16 0.291 98 22 60 41.3 2-5 0.9942
MIRU20 0.132 100 21 60 41.3 2-5 0.9942
MIRU24 0.107 102 22 58 40.0 2-5 0.9942
QUB11b 0.081 103 22 57 39.3 2-5 0.9942
MIRU27 0.067 105 21 55 37.9 2-4 0.9942
MIRU4 0.054 106 21 55 37.9 2-4 0.9942
MIRU23 0.041 107 21 54 37.2 2-4 0.9944
MIRU2 0.027 107 21 54 37.2 2-4 0.9942
ETR-B 0 107 21 54 37.2 2-4 0.9942
ETR-C 0 107 21 54 37.2 2-4 0.9942
25
Table 5. Demographic information of patients and genetic characteristics of the 47 MDR-MTB clustered isolates belonging to the CAS genotype.
Location Age Sex Year Categolya
SITb
MIRU-VNTRc RpoB in codon rpoB in nucleotide KatG inhA
127 Nepalgunj 24 F 2008 cat II failure 26 252235442248225153353543 R,I Gln 513 His, del: 514~516 del: 1296~1304 attcatgga Ser 315 Thr WT Cluster 1
130 Nepalgunj 30 F 2008 cat II failure 26 252235442248225153353543 R,I,E Gln 513 His, del: 514~516 del: 1296~1304 attcatgga Ser 315 Thr WT
342 Janakpur 28 M 2012 cat II failure 26 242236442248225153353743 R,I Ser 531 Leu C 1349 T Ser 315 Thr C -15 T Cluster 2
479 Nepalgunj 41 M 2012 cat II failure 26 242236442248225153353743 R,I,S,E - - Ser 315 Thr -
400 Pokhara 50 F 2008 cat II failure 471 242235442248225133353743 R,I,S,E Ser 531 Leu C 1349 T Ser 315 Thr WT
414 Pokhara 82 M 2011 cat II failure 471 242235442248225133353743 R,I,S,E del: 517 del: 1306~1308 cag Ser 315 Thr WT Cluster 3
417 Pokhara 33 M 2012 relapse 471 242235442248225133353743 R,I,S Ser 531 Leu C 1349 T Ser 315 Thr WT
491 Nepalgunj 34 F 2012 cat I failure 471 242235442248225133353743 R,I,S Asp 516 Tyr G 1303 T Ser 315 Thr WT
186 Dhangahi 22 F 2011 cat II failure 428 2422364422410225163323743 R,I Asp 516 Tyr G 1303 T Ser 315 Thr WT Cluster 4
516 Dhangahi 22 M 2012 cat II failure 428 2422364422410225163323743 R,I Asp 516 Tyr G 1303 T Ser 315 Thr WT
334 Janakpur 18 M 2009 cat II failure 356 242234442248225163353743 R,I,S,E Ser 531 Leu C 1349 T Ser 315 Thr WT Cluster 5
428 Bhairahawa 24 M 2008 cat II failure 356 242234442248225163353743 R,I,S Gln 513 Leu A 1295 T Ser 315 Thr WT
526 Kathmandu 40 F 2008 cat II failure 357 242234442238225163353743 R,I,S,E Asp 516 Val A 1304 T Ser 315 Thr WT
556 Kathmandu 34 M 2009 cat II failure 357 242234442238225163353743 R,I,E Asp 516 Phe G 1303 T, A 1304 T Ser 315 Thr WT Cluster 6
557 Kathmandu 40 M 2009 cat II failure 357 242234442238225163353743 R,I,E Asp 516 Phe G 1303 T, A 1304 T Ser 315 Thr WT
647 Kathmandu 24 M 2010 cat II failure 357 242235442238225163353743 R,I,S Asp 516 Val A 1304 T Ser 315 Thr WT Cluster 7
652 Kathmandu 35 M 2011 cat I failure 357 242235442238225163353743 R,I Asp 516 Phe G 1303 T, A 1304 T Ser 315 Thr WT
250 Kathmandu 36 M 2009 cat II failure 288 242235442248225183353743 R,I Ser 531 Leu C 1349 T Ser 315 Thr WT Cluster 8
617 Kathmandu 20 M 2010 cat II failure 288 242235442248225183353743 R,I,S Ser 531 Leu C 1349 T Ser 315 Thr WT
181 Dhangahi 31 F 2010 cat II failure 26 282226442238225163353843 R,I,S Ser 531 Leu C 1349 T Ser 315 Thr WT Cluster 9
477 Nepalgunj 62 M 2012 defaulter 26 282226442238225163353843 R,I WT none WT WT
184 Dhangahi 32 F 2010 cat II failure 1312 232236442247225153353743 R,I,S Ser 531 Leu C 1349 T Ser 315 Thr WT Cluster 10
488 Nepalgunj 25 M 2012 cat II failure 1312 232236442247225153353743 R,I Ser 531 Leu C 1349 T Ser 315 Thr WT
505 Dhangahi 32 M 2011 cat II failure 25 232236422248225153353543 R,I Leu 511 Pro T 1289 C Ser 315 Thr WT Cluster 11
547 Kathmandu 36 F 2009 cat II failure 25 232236422248225153353543 R,IS,E Leu 511 Pro T 1289 C Ser 315 Thr WT
117 Butwal 34 M 2011 cat II failure 26 242236442248425163344642 R,I,S,E Ser 531 Leu C 1349 T Ser 315 Thr WT Cluster 12
469 Nepalgunj 30 M 2009 cat II failure 26 242236442248425163344-42 R,I,S Ser 531 Leu C 1349 T Ser 315 Thr WT
421 Bhairahawa 61 M 2008 cat II failure 26 242236442248425183344742 R,I,S,E His 526 Asp C 1333 G Ser 315 Thr T -8 C
426 Bhairahawa 30 M 2009 cat II failure 26 242236442248425183344742 R,I,S,E His 526 Asp C 1333 G Ser 315 Thr T -8 C
439 Butwal 31 M 2012 cat II failure 26 242236442248425183344742 R,I His 526 Tyr C 1333 T Ser 315 Thr WT Cluster 13
536 Kathmandu 30 F 2009 cat II failure 26 242236442248425183344742 R,I,S,E His 526 Asp C 1333 G Ser 315 Thr WT
543 Kathmandu 73 M 2009 cat II failure 26 242236442248425183344742 R,I,S His 526 Asp C 1333 G Ser 315 Thr WT
641 Kathmandu 16 F 2010 cat I failure 26 242236442248425183344742 R,I,S Ser 531 Leu C 1349 T Ser 315 Thr WT
176 Mahendranagar 34 M 2009 cat II failure 486 222235442247425153343343 R,I,S,E Ser 531 Gln T 1348 C, C 1349 A Ser 315 Thr WT Cluster 14
177 Mahendranagar 55 F 2010 cat II failure 486 222235442247425153343343 R,I,S,E Ser 531 Gln T 1348 C, C 1349 A Ser 315 Thr WT
309 Kathmandu 18 F 2012 cat I failure 599 242226422238225163355723 R,I Ser 531 Leu C 1349 T Ser 315 Thr WT
460 Butwal 18 M 2012 cat I failure 599 242226422238225163355723 R,I,E Ser 531 Leu C 1349 T Ser 315 Thr WT Cluster 15
537 Kathmandu 25 M 2009 cat II failure 599 242226422238225163355723 R,I Ser 531 Leu C 1349 T Ser 315 Thr WT
628 Kathmandu 21 M 2010 cat I failure 599 242226422238225163355723 R,I,S,E Ser 531 Leu C 1349 T Ser 315 Thr WT
IDDST
resultsd
Mutations in drug resistance-associated geneseDemographic information of patients Genotype of isolates
26
317 Kathmandu 36 M 2012 cat I failure 599 242226422248225163355723 R,I,S Ser 531 Leu C 1349 T Ser 315 Thr WT Cluster 16
538 Kathmandu 16 F 2009 MDR contact 599 242226422248225163355723 R,I,S Ser 531 Leu C 1349 T Ser 315 Thr WT
394 Dharan 25 F 2011 new 22 242216442248225163453543 R,I,S,E Gln 513 Leu A 1295 T Ser 315 Thr WT
471 Nepalgunj 23 M 2010 cat II failure 22 242216442248225163453543 R,I,S,E Gln 513 Leu A 1295 T Ser 315 Thr WT Cluster 17
483 Nepalgunj 22 M 2012 cat I failure 22 242216442248225163453543 R,I,S,E Gln 513 Leu A 1295 T Ser 315 Thr WT
542 Kathmandu 63 M 2009 cat II failure 22 242216442248225163453543 R,I,S,E Gln 513 Leu A 1295 T Ser 315 Thr WT
171 Nepalgunj 20 M 2011 cat II failure 427 242234452247425163253723 R,I,S,E Ser 531 Leu C 1349 T Ser 315 Thr WT Cluster 18
492 Nepalgunj 28 M 2012 relapse 427 242234452247425163253723 R,I Ser 531 Leu C 1349 T Ser 315 Thr WT
a: CAT II failure; patients who were either smear positive relapse, chronic, MDR contact, relapse and treatment after default, CAT I failure; failure in new cases of smear positive pulmonary TB
b: SIT (Spoligo International types) were assigned by SITVITWEB database and MIRU-VNTR plus web tool
c: Order of loci; MIRU2, VNTR424, ETR-C, MIRU4, MIRU40, MIRU10, MIRU16, VNTR1955, MIRU20, QUB11b, ETR-A, QUB11a, VNTR2401, ETR-B, MIRU23, MIRU24, MIRU26, MIRU27, ETR-F, MIRU31,
VNTR3690, QUB26, QUB4156 and MIRU39
d: Drug susceptibility test results. Isolates showed resistance against R: rifampicin, I: isoniazid, S: streptomycin, E: ehambutol
e: Amino acid substitutions are shown in the codon number. Del: deletion, -: PCR failed, WT: wild type sequence. KatG Ser 315 Thr substitution was by katG G944C mutation.
27
Summary
Tuberculosis caused by Mycobacterium tuberculosis (MTB) poses a major public health
problem in Nepal. Although it has been reported as one of the dominant genotypes of MTB in
Nepal, little information on the Central Asian Strain (CAS) family is available, especially
isolates related to multidrug resistance (MDR) cases. Here, I aimed to elucidate the genetic and
epidemiological characteristics of MDR CAS isolates in Nepal. A total of 145 MDR CAS
isolates collected in Nepal from 2008 to 2013 were characterized by spoligotyping,
mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR)
analysis and drug resistance-associating gene sequencing. Spoligotyping analysis showed
CAS1_Delhi SIT26 as predominant (60/145, 41.4%). However, by combining spoligotyping
and MIRU-VNTR typing, I was able to successfully discriminate all 145 isolates into 116
different types including 18 clusters with 47 isolates (clustering rate: 32.4%). About a half of
these isolates shared the same genetic and geographical characteristics with other isolates in
each cluster, and some of them shared rare point mutations in rpoB that were thought to
associate with rifampicin resistance. Although the obtained data showed little evidence that
large outbreaks of MDR-TB by CAS family occurred in Nepal, they strongly suggested several
MDR-MTB transmission cases. Therefore, I believe the proposed 15 loci MIRU-VNTR typing
scheme is well suited to assess the population structure of the MDR CAS family and trace back
the transmission dynamics in Nepal.
28
Chapter II
Characterization of genetic diversity of multidrug resistant Mycobacterium tuberculosis
Central Asian Strain isolates from Nepal and comparison with neighboring countries
Introduction
MTB comprises of four major lineages, which can be differentiated based on specific
genetic markers and geographical areas (11, 20, 21, 22). Lineage 3 including the spoligotype-
defined Central Asian Strain (CAS) family is predominant genogroup and spreading in South
Asian countries including India, Pakistan and Nepal (1, 2, 24, 29, 41, 43, 44, 48). In
spoligotyping, CAS family is characterized by the deletion of spacers 4-7 and 23-34 in the direct
repeat locus and divided into more than 25 subfamilies (36). Some studies on genotyping from
Nepal, Northern India and Pakistan revealed that the emergence of MDR CAS family is posing
a serious threat to TB control in South Asian countries (1, 41, 43, 44).
The main purpose of this study was to better understand the genetic diversity, transmission
dynamics and evolutionary relationships of MDR CAS family isolates, especially the major
clade of CAS1_Dehli SIT26, circulating in Nepal and neighboring countries. For this purpose,
I constructed a phylogenetic tree and minimum spanning trees (MSTs) to gain insight into the
genetic characteristics of MDR CAS family by using molecular genotyping tools such as
spoligotyping and MIRU-VNTR typing.
29
Materials and Methods
Sample collection and drug susceptibility test
A total of 145 MDR-TB CAS family isolates were purposively collected from April 2008 to
March 2013 by German Nepal Tuberculosis Project (GENETUP) using a decentralized Nepal
Tuberculosis Programme (NTP) network (Figure 1). Drug susceptibility test (DST) was carried
out using the conventional proportion method for all first line anti-TB drugs (INH, RIF, STR,
EMB) as described previously (54). Furthermore, I reviewed publications on MDR-TB CAS
family from neighboring countries, India and Pakistan (1, 2, 9), to collect the genotyping data
to compare their genetic diversity with Nepalese samples.
DNA extraction
DNA was extracted from mycobacterial culture. Briefly, colonies from positive LJ cultures were
suspended in 300 µl of DNA free distilled water and heated for 20 min at 95 oC. The heated
samples were incubated in an ultra-sonic water bath for 15 min, centrifuged for 5 min at 10,000
g and then supernatant containing bacterial DNA was used for further molecular analysis.
Spoligotyping and MIRU-VNTR typing
All isolates were analyzed by spoligotyping, as described by Kamerbeek et al. (28). Spoligo-
international type (SIT) were determined using SITVITWEB database (http://www.pasteur-
guadeloupe.fr:8081/SITVIT_ONLINE/) and MIRU-VNTRplus (15). MIRU-VNTR typing was
carried out by amplifying 24 loci that included 12 MIRU loci (MIRU2, MIRU4, MIRU10,
MIRU16, MIRU20, MIRU23, MIRU24, MIRU26, MIRU27, MIRU31, MIRU39 and MIRU40),
4 exact tandem repeats (ETR) loci (ETR-A, ETR-B, ETR-C and ETR-F), 4 Queens University
Belfast (QUB) loci (QUB11a, QUB11b, QUB26 and QUB4156) and 4 VNTR loci (VNTR424,
VNTR1955, VNTR2401 and VNTR3690) as described by Supply et al. (46). The copy number
of repeats for each locus was determined based on the allelic table as given by Supply et al.
(46).
Data management and analysis
Hunter Gaston discriminatory indices (HGDI) were calculated to determine the diversity of
each locus (27). The discriminatory power of each locus was determined as high (HGDI>0.6),
moderate (0.3≤ HGDI ≤0.6) and poor (HGDI<0.3) as suggested by Sola et al. (45). A cluster
was defined as two or more isolates sharing the identical spoligotype and MIRU-VNTR patterns
30
and clustering rate was calculated using the formula: number of clustered isolates / total number
of isolates (23). Phylogenetic tree was constructed by unweighted pair group method with
arithmetic averages (UPGMA) using an online MIRU-VNTRplus web based application
[http://www.miru-vntrplus.org] (51).
Minimum spanning trees (MSTs)
MSTs were generated by all the obtained MIRU-VNTR data (n=145) to analyze the
evolutionary relationship among the Nepalese isolates using BioNumerics software version 6.6
(Applied Maths, Sint-Martens-Latem, Belgium) with additional information of spoligotype
(SIT), isolated reagions or isolated years. For the comparison with neighboring-countries’
isolates of India (Mumbai n=16) (9) and Pakistan (n=62) (2), MSTs were drawn with the MIRU-
VNTR patterns of CAS1_Delhi SIT26 subfamily of Nepal (n=60), since the data of other
subfamilies were not available from those neighboring countries.
31
Results
MIRU-VNTR analysis
A combination of spoligotyping and 24-loci MIRU-VNTR analysis could differentiate the 145
MDR CAS family isolates into 116 different patterns as shown in Chapter I. The biggest
spoligotype cluster CAS1_Delhi SIT26, which consisted of 60 isolates, was further
differentiated into 49 MIRU-VNTR types, i.e. five clusters composed of 14 isolates and
remaining isolates with unique patterns. The clustering rate was 23.3% and the biggest cluster
consisted of 6 isolates whereas other four clusters were by 2 isolates (Figure 4). Individual
allelic diversity of each MIRU-VNTR locus of the 60 isolates was estimated by using HGDI
(Table 6). The discriminatory powers of three out of 24 loci (QUB26, MIRU26, VNTR424)
were found to be highly discriminatory, 8 loci (MIRU10, MIRU31, VNTR3690, MIRU39,
VNTR 2401, MIRU40, QUB4156 and ETR-F) were moderately discriminatory and remaining
13 loci (QUB11a, MIRU16, ETRA, MIRU4, VNTR1955, MIRU27, QUB11, MIRU20, MIRU2,
ETR-C, MIRU23, MIRU24 and ETR-B) were poorly discriminatory. I also calculated and
compared the allelic diversity of MIRU-VNTR of Nepalese CAS1_Delhi SIT26 isolates with
those reported from neighboring countries, Pakistan and India, in Table 7. Allelic diversity,
HGDI, of each MIRU-VNTR loci were different among countries and those values of Nepalese
isolates tended to lower than other countries.
MST analysis
MSTs were drawn with the 24-loci MIRU-VNTR typing data of 145 MDR CAS family isolates
from Nepal with following variables; SIT, isolated region and year as shown in Figures 5, 6 and
7, respectively. CAS1_Delhi SIT26 subfamily isolates were more diverse than other SITs and
formed the biggest cluster (Figure 5). No specific regional clusters or yearly trends were
observed by the region wise (Figure 6) and year wise (Figure 7) MST analyses. To compare the
Nepalese CAS1_Delhi SIT26 isolates with neighboring countries, MST analysis were carried
out with 15-loci MIRU-VNTR for Pakistani isolates and 12-loci MIRU-VNTR for Indian
isolates (Figure 8) depending on the data availability. An Indian isolate shared identical MIRU-
VNTR pattern to Nepalese strains, while no Pakistani strain shared the same pattern with
32
Nepalese. Pakistani isolates showed higher genetic diversity than Nepalese isolates.
33
Discussion
CAS family is reported to be highly prevalent among MTB strains in Nepal and
neighboring countries like India (46-68%) (9, 44) and Pakistan (77%) (57). These data support
that the CAS family is a dominate lineage of MTB in certain regions of Indian subcontinent.
However, recent studies demostrated that this strain is also circulating among the local
population in other geographical areas like Sudan (49%), Ethiopia (38.9%), Saudi Arabia
(26.4%), Iraq (24%), Iran (24%) and Egypt (6.1%) (3, 17, 26, 31, 40, 47). Geographically, Nepal
is located between two high TB burden countries, India and China which contribute one third
of world’s TB cases (53, 56). CAS family is one of the dominant strains in northern part of
India whereas Indo-Oceanic family is dominant in the southern and central India (34, 42, 43).
Nepal has open borders with India and Nepalese citizens visit the bordering area of northern
India for different purposes including work, pilgrimage and education. This contact could
provide an opportunity of transmission for CAS family. A similar study in Tibet also linked the
spread of CAS family to trade, tourism and migration from India (18). Current study found
clustering rates of 32.4% in total 145 MDR CAS isolates and 23.3% in subcluster SIT26
suggesting a possible ongoing transmission. Similar to our findings, high clustering rate among
the CAS family have been reported from Saudi Arabia (22.2%) among the foreign-born
nationals, South East Asia (35.3%), Indian sub-continent (18.4%) and West/Horn of Africa
(10.2%) (3). Therefore, there is urgent need for effective control strategies to cut the MTB
transmission chain between Nepal and neighboring countries.
MSTs were constructed to understand the genetic characteristics and evolutional
relationship of 145 MDR CAS family isolates from Nepal (Figure 5, 6 and 7). Overall analysis
of SIT, region wise and year wise MSTs strongly suggested that the Nepalese MDR CAS family
isolates were genetically diverse and most probably, MDR strains emerged in each patient
independently due to a selective pressure of anti-TB drugs. On the other hand, only little
evidences of possible MDR-MTB transmissions between individuals in the same cluster were
shown (Table 5). I could not trace out any personal contacts of patients who were in the MDR-
MTB transmission-suggested cases, and it ought to be indicating the existence of a common
source. It could be speculated that a trans-border visitor might be a missing link of the
34
transmission chain since a large number of TB patients from northern India have been travelling
to Nepal for cheaper TB treatment (35, 37).
In this study, spoligotyping only could not precisely discriminate the pattern of diverse CAS
family in Nepal. By a spoligotyping, CAS1_Delhi SIT26 comprised of 60 isolates was the most
predominant subfamily among 145 CAS family strains (Figure 4). Thus, the 60 MDR
CAS1_Delhi SIT26 strains were subjected to further analysis using the 24-loci MIRU-VNTR
typing to see the genetic diversity and were successfully discriminated into 49 different MIRU
patterns including 5 clusters. The largest MIRU-VNTR-defined cluster composed of six isolates
and these isolates were from three different geographic locations (Table 5). This strain type may
be one of the dominant strains of CAS family in Nepal. Isolates belonging to CAS1_Delhi
SIT26 isolates exhibited diverse MIRU-VNTR patterns in MST (Figure 5). This observation
may suggest that the strain might have adapted to Nepalese people over a long period with
acquiring genetic diversity and resulted in the establishment as the dominant strain among
Nepalese population (35). Since the CAS1_Delhi SIT26 spoligotype has the basic pattern of
the CAS family (spacers 4-7 and 23-34 deleted), this strain should be the parental type that must
have a longer history than others SITs, which evolved later on.
I also compared allelic diversity of MIRU-VNTR of CAS1_Delhi SIT26 with those
published from neighboring countries. In our study MIRU alleles (QUB11a, MIRU16, ETR-A,
MIRU4, VNTR1955, MIRU27, QUB11b, MIRU20, MIRU2, ETR-C, MIRU23, MIRU24 and
ETR-B) were highly conserved and showed poor discriminatory power (HGDI<0.3). However,
the same locus had different allelic diversity in surrounding countries and Nepalese HGDIs
tended to lower than other countries (Table 7). This finding is consisent with the other reports
that suggest difference in allelic diversity due to difference in geographical region and genetic
diversity of MTB families (1, 2). Since, I was interested to understand the relationship between
Nepalese isolates and other South Asian countries isolates, I constructed MSTs of our MDR
CAS1_Delhi SIT26 isolates together with those isolates previously reported from surrounding
countries, India and Pakistan (2, 9) (Figure 8). The topologies of MSTs based on the data of
three countries suggested that clones of CAS1_Delhi SIT26 in Pakistan were highly diverse
and genetically distant from Nepalese when compared with Indian. As expected, an Indian
35
isolate shared the same MIRU-VNTR pattern with a Nepalese MDR CAS1_Delhi SIT26 cluster.
I therefore elucidate that the existence of evolutionary relationship and possible trans-border
transmission between Nepal and India. The present study findings support Wirth et al. (52) who
speculated that CAS family might have emerged over 9,000 years ago in the Indian subcontinent.
Therefore, further studies of MDR-MTB CAS family isolates of Nepal and surrounding
countries will be needed to elucidate the epidemiological link, transmission chains and also its
evolutionary history using validated molecular approaches e.g. whole genome sequence
analysis.
36
Table 6: Individual and Cumulative HGDI of CAS1_ Delhi (SIT26) (N=60)
VNTR alias Individual
HGDI
No. of
clusters
No. of
clustered
isolates
Clustering
rate (%)
No. of
isolates in
each cluster
Cumulative
HGDI
QUB26 0.6712 0.6712
MIRU26 0.6292 9 58 96.6 2-19 0.8965
VNTR424 0.6185 10 44 73.3 2-8 0.9595
MIRU10 0.5330 11 40 66.6 2-7 0.9728
MIRU31 0.5105 11 35 58.3 2-7 0.9779
VNTR3690 0.4920 10 31 51.6 2-7 0.9810
MIRU39 0.4889 10 31 51.6 2-7 0.9810
VNTR2401 0.4004 9 28 46.6 2-7 0.9841
MIRU40 0.3973 9 25 41.6 2-6 0.9866
QUB4156 0.3195 9 24 40.0 2-6 0.9882
ETR-F 0.3143 9 24 40.0 2-6 0.9882
QUB11a 0.2908 8 21 35.0 2-6 0.9897
MIRU16 0.2800 8 21 35.0 2-6 0.9897
ETR-A 0.2759 8 21 35.0 2-6 0.9897
MIRU4 0.1536 8 21 35.0 2-6 0.9897
VNTR1955 0.1525 7 19 31.6 2-6 0.9902
MIRU27 0.0937 7 19 31.6 2-6 0.9902
QUB11b 0.0317 7 19 31.6 2-6 0.9902
MIRU20 0.0317 6 17 28.3 2-6 0.9907
MIRU2 0 6 17 28.3 2-6 0.9907
ETR-C 0 6 17 28.3 2-6 0.9907
MIRU23 0 6 17 28.3 2-6 0.9907
MIRU24 0 6 17 28.3 2-6 0.9907
ETR-B 0 6 17 28.3 2-6 0.9907
37
Figure 4: 24-locus MIRU-VNTR based dendrogram of 60 MDR CAS1_Delhi (SIT26) isolates
from Nepal. (A) The dendrogram was generated by UPGMA [www.miru-vntrplus.org]. (B)
Strain identification number. (C) MIRU-VNTR results of 24 loci. The order of loci is as follows,
left to right: MIRU2, VNTR424, ETR-C, MIRU4, MIRU40, MIRU10, MIRU16, VNTR 1955,
MIRU20, QUB11b, ETR-A, QUB11a, VNTR 2401, ETR-B, MIRU23, MIRU24, MIRU26,
MIRU27, ETR-F, MIRU31, VNTR 3690, QUB26, QUB4156 and MIRU39. (D) Spoligotype
pattern. (E) SIT (Spoligo-international type) number. (F) Clade annotated by SITVIT database.
Isolates clusters (identical patterns of MIRU-VNTR) are enclosed in open boxes.
38
Table 7: Allelic diversity of MIRU-VNTR loci in MDR CAS family (SIT26) strains from
Nepal and neighboring countries.
VNTR alias VNTR
locus
Nepal (N=60)
(current
study)
Pakistan
(N=62) [1]
Pakistan
(N=20) [2]
India
(Munbai)
(N=16) [9]
QUB26 4052 0.671 - 0.821 -
MIRU26 2996 0.629 0.808 0.721 0.766
VNTR424 424 0.618 - 0.721 -
MIRU10 960 0.533 0.753 0.70 0.775
MIRU31 3192 0.510 0.758 0.668 0.241
VNTR3690 3690 0.492 - 0.568 -
MIRU39 4348 0.488 0.715 - 0.125
VNTR2401 2401 0.400 - 0.568 -
MIRU40 802 0.397 0.715 0.468 0.675
QUB4156 4156 0.319 - 0.594 -
ETR-F 3293 0.314 - - -
QUB11a 2163a 0.290 - - -
MIRU16 1644 0.280 0.778 0.531 0.575
ETR-A 2165 0.275 - 0.531 -
MIRU4 580 0.153 0.287 0 0.125
VNTR1955 1955 0.152 - 0.194 -
MIRU27 3007 0.093 0.750 - 0
QUB11b 2163b 0.031 - 0.268 -
MIRU20 2059 0.031 0.670 - 0
MIRU2 154 0 0.546 - 0.125
ETR-C 577 0 - 0.278 -
MIRU23 2531 0 0.546 - 0.233
MIRU24 2687 0 0.521 - 0.125
ETR-B 2461 0 - - -
N: Number of MDR CAS family (SIT26) isolates
-: Not reported
[1] Ali A et al. (2007)
[2] Ali A et al. (2014)
[9] Chatterje and Mistry (2012)
39
Figure 5: Minimum spanning tree (MST) constructed based on diversity of the 24 loci MIRU-
VNTR results of 145 MDR MTB CAS family. The size of circle is proportional to the total
number of isolates representing unique isolates (smaller nodes) and clustered isolates (bigger
nodes). The color of the circles indicates the CAS family belongs to particular Spoligotype-
International type (SIT). The MST connects each sub-family based on degree of changes
required from one allele to another; solid lines (1or 2 or 3 changes), gray dashed lines (4) and
gray dotted lines (5 or more changes).
Lines denotes the number of allele changes
1 2 3 4 5 ................
40
Figure 6: Minimum spanning tree (MST) constructed based on region wise distribution of 145
MDR MTB CAS family. The size of circle is proportional to the total number of isolates
representing unique isolates (smaller nodes) and clustered isolates (bigger nodes). The color of
the circles indicates the CAS family belongs to regionwise. The MST connects each sub-family
based on degree of changes required from one allele to another; solid lines (1or 2 or 3 changes),
gray dashed lines (4) and gray dotted lines (5 or more changes).
41
Figure 7: Minimum spanning tree (MST) constructed based on year wise isolation MIRU-
VNTR results of 145 MDR MTB CAS family. The size of circle is proportional to the total
number of isolates representing unique isolates (smaller nodes) and clustered isolates (bigger
nodes). The color of the circles indicates the CAS family belongs to yearwise. The MST
connects each sub-family based on degree of changes required from one allele to another; solid
lines (1or 2 or 3 changes), gray dashed lines (4) and gray dotted lines (5 or more changes).
42
Figure 8: MIRU-VNTR based minimum spanning tree (MST) was constructed to trace out the
evolutionary relationship of MDR MTB CAS family (SIT26) of Nepal and neighboring
countries (India and Pakistan). (a) Comparative MST based on 15 loci between Pakistan and
Nepal (b) Comparative MST based on 12 loci between India and Nepal. The size of circle is
proportional to the total number of isolates; unique isolates (smaller nodes) and clustered
isolates (bigger nodes). The color of nodes indicates corresponding country of origin of isolates.
Pakistan (Ali A et al.2014)
Nepal (In this study)
India (Chatterjee and Mistry 2012)
Nepal (In this study)
a.15 loci MIRU-VNTR b.12 loci MIRU-VNTR
43
Summary
Multidrug-resistant Tuberculosis (MDR-TB) is an emerging threat for successful TB
control in Indian subcontinent region. Central Asian strain (CAS) family has been reported as
one of the dominant families contributing to MDR-TB in South Asia including Nepal, India
and Pakistan. The aim of this study was to better understand the genetic characteristics of MDR
CAS family isolates circulating in Nepal, as well as in neighboring countries.
A total of 145 MDR-TB CAS family isolates collected in Nepal from 2008 to 2013 were
analyzed by spoligotyping and mycobacterial interspersed repetitive units-variable number of
tandem repeats (MIRU-VNTR) analysis. I also compared these data with published data from
India and Pakistan to investigate possible epidemiological link through construction of
Minimum spanning tree (MST). Spoligotyping analysis exhibited CAS1_Delhi SIT26 (n=60)
was the predominant lineage among MDR CAS family in all three countries. However, by
combining two genotyping methods, spoligotyping and MIRU-VNTR, 60 isolates were further
discrimnated into 49 different types and 5 clusters. These clusters composed of 14 isolates with
clustering rate (23.3%), suggesting ongoing transmissions. Based on MST data from
neighboring countries, I elucidated an evolutionary relationship between the two countries,
Nepal and India, which could be explained by their open borders. This study identified the
evolutionary relationships among MDR CAS1_Delhi subfamily isolates from Nepal and those
from neighboring countries.
44
Conclusion
Central Asian strain (CAS) family is one of the dominant MTB families among MDR-MTB,
which is emerging in South Asian countries including Nepal. To date, there are only limited
studies have been reported and little is known about the genetic diversity of MDR-MTB CAS
family in Nepal and neighboring countries. Therefore, the present study was necessary to gain
insight into the characteristics of MDR CAS family circulating in Nepal and neighboring
countries like India and Pakistan.
In this regard, in chapter I, I investigated the genetic characteristics of MDR-MTB CAS
family isolates from Nepal by applying three molecular approaches, i.e. Spoligotyping, 24-loci
MIRU-VNTR analysis and drug resistance-associating genes (rpoB, KatG and the inhA
promoter region) analysis. Those molecular analyses demonstrated that the MDR CAS family
isolates were highly diverse in Nepal, which suggested that the bacteria progressively acquired
drug resistance and ultimately became MDR in each patient. Nonetheless, the genetic and
epidemiological characteristics of some clustered isolates showed the evidence of actual MDR-
MTB transmission. A large MDR-TB outbreak would be more likely to occur among the
Nepalese population if transmission trends observed in the present study grew out of control.
Thus, our results highlight the importance of laboratory diagnosis of TB, intensified finding of
cases and timely and appropriate treatment of TB patients to cut the transmission chain.
Therefore, I believe the proposed 15 loci MIRU-VNTR typing scheme will be well suited to
assess the population structure of the MDR CAS family and trace back the transmission
dynamics in Nepal.
In Chapter II, I elucidated the genetic characteristics and evolutionary relationships of
predominant MDR CAS subfamily isolates circulating in Nepal and neighboring countries by
using molecular approach. I found that CAS1_Delhi SIT26 was predominant among MDR-
MTB CAS family in South Asia, and widely spread throughout the Nepalese population with
higher diversity than other subfamilies. On the contrary, its diversity in Nepal was relatively
low in the comparison with neighboring countries, India and Pakistan, suggesting a shorter
endemicity history than those countries. In MST analysis, isolates of CAS1_Delhi SIT26 in
45
Pakistan were highly diverse and genetically distant from Nepalese when compared with Indian.
Indian CAS1_Delhi SIT26 family had similar MIRU-VNTR patterns to Nepalese strains with
one isolate shared the identical pattern with a Nepalese isolate cluster. Nepal has an open border
with India, and both Nepalese and Indian citizens often visit the bordering area for work,
pilgrimage and education. This regular interaction may open an opportunity for direct
transmission of the CAS family MTB between the Indian and Nepalese populations.
I believe that the findings of my study on the genetic diversity, epidemiological
characteristics and evolutionary relationships have extended our understandings on the
predominant MDR CAS family genotypes from Nepal and neighboring countries. It is foreseen
that the results from the present study will contribute to improve epidemiological surveillance,
which in turn could lead to implementation of more effective control measures against the
spread of MDR-MTB in Nepal and surrounding countries. The information gained by genetic
diversity of MDR CAS family isolates from the present study and comparison with neighboring
countries may provide base line information for future molecular epidemiological studies and
will also be helpful for assessments of TB Control programs in their respective countries.
46
ACKNOWLEDGEMENTS
It is my immense pleasure to express heartfelt appreciation and an enormous debt to my
respected supervisor Prof. Yasuhiko Suzuki from Division of Bioresources, Hokkaido
University Research Center for Zoonosis Control, Sapporo, Japan, for providing an excellent
opportunity for PhD study under his excellent supervision. His outstanding mentorship,
continuous support and valuable suggestions were extremely helpful to enhance my knowledge
and understanding in the field of tuberculosis (TB) research during my study.
I would also like to especially express my sincere and profound gratitude, and earnest
compliment to Assoc. Prof. Chie Nakajima, Division of Bioresources, Hokkaido University
Research Center for Zoonosis Control, Sapporo, Japan, for her invaluable guidance, outstanding
inspiration, tremendous support and encouragement to widen my knowledge and understanding
of TB and the genetic analyses of the isolates. Her guidance was very instrumental in planning
of the research, conducting the experiments and preparation of manuscript
I would like to express my sincere gratitude to Prof. Hideaki Higashi and Assoc. Prof.
Norikazu Isoda for their valuable suggestions, guidance and inputs in my TB research that was
extremely helpful to carry out my proposed research successfully. I am highly thankful to all
the faculties from Hokkaido University Research Center for Zoonosis control and Graduate
School of Veterinary Medicine for their kind support, guidance and recommendations in my
research as well as for their support to complete the required credit courses during my PhD
training here at Hokkaido University.
I am highly grateful to Mr. Bhagwan Maharjan and all members of German Nepal
Tuberculosis Project (GENETUP), Kathmandu, Nepal for their enormous help with collections,
processing and providing the DNA samples extracted from isolates of M. tuberculosis collected
from human TB patients of Nepal for my entire PhD research works. Everyone at GENETUP
were so welcoming and they were always ready to help with my research. I am also highly
indebted to Dr. Basu Dev Pandey from Everest International Clinic and Research Center, Nepal
for his genuine cooperation and encouragement throughout my study.
My especially thanks go to Dr. Ajay Poudel (Chitwan Medical College and teaching Hospital,
Nepal), Mr. Jeewan Thapa (Division of Bioresources, CZC), Dr. Hassan Mahmoud Diab (South
47
Valley University, Egypt), Mr. Eddie Solo (University Teaching Hospital, Zambia) supports
during the research experiments as well as preparation of the manuscript. I would also like to
thanks to Ms. Yukari Fukushima and Ms. Haruka Suzuki from Division of Bioresources,
Hokkaido University Research Center for Zoonosis control for their technical support during
the genotyping experiments.
I would like to express my heartfelt thanks to Dr. Kanjana Changkaew, Dr. Siriporn Kongsoi,
Dr. Ruchirada Changkwanyeun, Dr. Marvin Ardeza Villanueza, Dr. Tomoyuki Yamaguchi, Ms.
Nan Aye Thida Oo, Ms. Lai Lai San, Ms. Charitha Mendis, Mr. Jong-Hoon Park, Mr. Yuko
Ouchi, Mr. Kentaro Koide, Ms. Dipti Shrestha, Ms. Ruttana Pachanon, Mr. Thoka Flav
Kapalamula, Ms. Mwangala Lonah Akapelwa, Ms. Risa Tsunoda, Ms. Conschilliah Menda, Dr.
Yayoi Kameda, Ms. Miki Nakagawa, Ms. Janisara Rudeeaneksin, Dr. Fuangfa Utrarakij, Dr.
Nipawit Karnbunchob, Mr. Alex Samuel Kiarie Gaithuma and the rest of the members of CZC
for creation my four years stay in Sapporo, Japan fruitful, enjoyable and unforgettable moments
through out of my life as well as for their kind support and constant inspirations during my
research and studies. I am also like to conveying especially thanks our division secretaries Ms.
Midori Yoshida and Ms.Yuko Hidaka for their countless help me in many administrative works
and additional support in my difficult situation during my stay in Sapporo, Japan.
Respectfully, I would like to jot down my deepest thanks to all my senior teachers and friends
especially to Dr. Sher Bahadur Pun (Sukraraj Tropical and Infectious Disease Hospital,
Kathmandu, Nepal), Dr. Kishor Pandey (Nepal Academy of Science and Technology, Lalitpur,
Nepal), Dr.Sarad Paudel (Department of Cell physiology, Hokkaido University, Sapporo,
Japan) and Mr.Rabin Kadariya (Laboratory of Wildlife Biology and Medicine, Hokkaido
University, Sapporo, Japan) for providing me necessary assistance and invaluable suggestions
during the study period.
This research study was supported by the Leading Program at Hokkaido University, Graduate
School of Veterinary Medicine, Japan “Fostering Global Leaders in Veterinary Science toward
Contributing to ‘One Health’” from Ministry of Education, Culture, Sports, Science and
Technology, Japan (MEXT), U.S.-Japan Cooperative Medical Science Programs from Japan
Agency for Medical Research and development (AMED), Japanese Society for the Promotion
48
of Science (JSPS) ROMPAKU Program, MEXT/JSPS KAKENHI and Research Program on
Emerging and Re-emerging Infectious Diseases from AMED.
Finally, I am greatly obliged to my parents, wife and lovely (Priya/Priyan) without whose
constant inspiration and unconditional support, this work would not have been completed.
49
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