GENETIC DIVERSITY AND MOLECULAR BASIS OF
ANTIMICROBIAL RESISTANCE OF MYCOBACTERIUM
TUBERCULOSIS STRAINS FROM PAKISTAN
By
ASHO ALI
A thesis submitted in partial fulfilment of the requirements for the degree of PhD in Health Sciences
in the discipline of Microbiology
Department of Pathology and Microbiology Faculty of Health Sciences
Aga Khan University Karachi, Pakistan
2009
© Copyright
ii
Genetic Diversity and Molecular basis of Antimicrobial
resistance of Mycobacterium tuberculosis strains from Pakistan
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Health Sciences) in the discipline of Microbiology
Members of the Thesis Defense Panel appointed to examine the thesis of
Asho Ali
find it satisfactory and recommended that it be accepted
_____________________________ Dr. Rumina Hassan (Thesis Supervisor)
_____________________________ Dr El-Nasir Lalani (Internal Examiner)
_____________________________ Dr. Rana Muzaffar
(External Examiner)
_____________________________ Dr. Anwar-ul-Hassan Gilani
(Convener, Thesis Defense Panel)
Department of Pathology and Microbiology
Aga Khan University Karachi, Pakistan
March 11, 2009
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This thesis is dedicated to my parents!
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Abstract
Pakistan ranks 8th amongst the 22 high burden tuberculosis (TB) disease countries
with an estimated incidence rate of 181/100,000 population. The high incidence of
tuberculosis in Pakistan is further compounded by the increasing emergence of drug
resistant strains including multi-drug resistant (MDR). Prevalence of MDR-TB in
Pakistan has been shown to be between 2-4% in the untreated patients, while the global
prevalence of MDR is estimated at 3%.
Molecular epidemiological studies, based on the assumption that patients infected
with clustered strains are epidemiologically linked, have helped understand the
transmission dynamics of disease. It has also helped to investigate the basis of variation
in Mycobacterium tuberculosis (MTB) strains, differences in transmission, severity of
disease or drug resistance mechanisms in a defined geographical location. This is in turn
helpful in developing strategies for the treatment and prevention of the disease including
MDR. Molecular epidemiological data using spoligotyping available from Pakistan
shows a predominance of the Central Asian Strain1 (CAS1) (39%), and Beijing strains
(6%). Beijing strains have been shown an association with MDR. Although CAS1 strains
have not shown an association with MDR, about forty percent of total CAS1 strains were
comprised of MDR strains. The data about the types and frequency of drug resistance
gene mutations within these strains is limited.
The overall aims of this study were to explore genetic diversity amongst the
predominant genogroup of Mycobacterium tuberculosis from the country as well as to
investigate genetic basis of drug resistance amongst these predominant MTB strains. In
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this study variable number of tandem repeat mycobacterial interspersed repetitive unit
(VNTR-MIRU) and IS6110 based restriction fragment length polymorphism (IS6110-
RFLP) was used to study the relationship within CAS1 strains. Knowledge of type and
frequency of mutations amongst prevalent genogroup has been shown to be essential for
the development of appropriate tools for early diagnosis and control of MDR-TB strains.
There is limited information on mutations leading to drug resistance within MTB strains
from the country. Therefore in this study prevalent mutation in the rpoB, katG and inhA
genes for rifampicin (RMP) and isoniazid (INH) resistance were also investigated in
MDR strains of predominant genogroups.
Twelve loci based VNTR-MIRU typing showed highly diverse profile of 367
MTB strains. Of the 178 CAS1 strains studied only 34 (19 %) clustered into groups
based on MIRU profiles, while all 189 ‘unique’ spoligotypes studied had non-matching
MIRU profiles and therefore remained un-clustered. The MIRU-VNTR data shows a
close relationship (70%) within the prevalent CAS1 strains. Therefore it is proposed that
in a region where CAS1 family strains are prevalent most discriminatory MIRU loci 26,
31, 16, 10, 27, 39 and 40, could be used for differentiation and estimation of the
phylogenetic relatedness of Mycobacterium tuberculosis. IS6110-RFLP typing of 78
strains (43 CAS1 and 35 ‘unique’ spoligotypes strains) resulted in 73 different RFLP
types. One cluster of two unique strains, with single copy of IS6110 was identified; the
remaining seventy-two strains revealed unique RFLP patterns.
The most common mutations determined in MDR strains were in codons 531
(60%), 526 (23%) and 516 (5%) of rpoB gene by sequencing, while probe based assay
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detected 44% and 21% mutations at codon 531 and 526 respectively. Occurrence of
mutation at codon 526 as well as concurrent mutation in rpoB gene was significantly
higher in CAS1 than Beijing and other un-clustered strains tested. Sixty three percent of
62 MDR isolates showed mutation at codon 315 by sequencing while by probe based
assay sixty percent of resistance was detected. CAS1 family strains exhibited higher rate
of mutation at codon 315 as compared with Beijing.
Multi-drug resistant CAS1 strains are more prone to develop resistance against
RMP and INH through mutations at codon 526 of rpoB gene and codon 315 of katG gene
respectively than non-CAS1 MDR strains. 67% of RMP and INH resistance within MDR
CAS1 strains could be determined by detecting mutations at only three codons 526 and
531 of rpoB and 315 of katG gene.
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List of abbreviations
AFB Acid Fast Bacilli ATT Anti-tuberculosis therapy BCG Bacillus Calmette-Guerin bp base pairs CAS1 Central Asian Strain1 DMSO Di methyl sulf oxide DNA Deoxyribonucleic Acid DOTS Direct Observed Treatment Strategy DR Direct Repeat E Ethambutol ETR Exact Tandem Repeats FRET Fluorescence Resonance Energy Transfer HGDI Hunter Gaston Discriminatory Index HBC High Burden Country IDU Injection Drug Users HIV Human Immunodeficiency Virus INH Isoniazid IS Insertion Sequence IUATLD International Union against Tuberculosis and Lung Diseases LiPA Line Probe Assay LJ Lowenstein Jensen LSP Large Sequence Polymorphism MDR Multi Drug Resistance MDR-TB Multi Drug Resistance Tuberculosis MTB Mycobacterium tuberculosis MIRU Mycobacterial Interspersed Repetitive Unit MTC Mycobacterium tuberculosis complex NTP National Tuberculosis control Program PCR Polymerase Chain Reaction PYZ Pyrazinamide RFLP Restriction Fragment Length Polymorphism RMP Rifampicin SNP Single Nucleotide Polymorphism SPSS Special Program for Social Sciences Software TB Tuberculosis UPGMA Un-weighted pair group using arithmetic averages VNTR Variable Number of Tandem Repeat WHO World Health Organization
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ACKNOWLEDGMENT
I would like to express my genuine gratitude and sincere thanks to all those who
have helped and encouraged me during this thesis work especially;
Dr Rumina Hasan, my supervisor, who from the first day of her involvement in this
project invested all her time and effort until the day this thesis work became a reality. Her
excellent scientific guidance, constant support and encouragement throughout the thesis
work brought it to completion.
Dr Zahra Hasan, my co-supervisor for making exploration of genomic world of
Mycobacterium tuberculosis possible for me. Her endless support, enduring
encouragement and kindness led me through this thesis.
Dr Rabia Hussain, my thesis committee member, for her expert advisement and
scientific criticism during initial phase of the study.
Dr Rehana Siddiqui, my thesis committee member, for helping me out in statistical
calculations of samples and guidance time to time during this thesis period.
Dr Tariq Moatter, for all his guidance in making detection of drug resistance possible
through real time PCR.
Dr Anwar Ali Siddiqui, Associate Dean Research for his sincere support through out this
thesis period.
Dr Ruth McNerney, from The London School of Hygiene and Tropical Medicine, UK
for guidance in data analysis of MIRU-VNTR typing.
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Dr Kristin Kremer from Mycobacteria Reference Unit, Diagnostic Laboratory, The
Netherlands for providing reference strains of Mycobacterium tuberculosis for RFLP.
Ramona Petersson and Solomon Ghebremichael from Swedish Institute for Infectious
Disease Control, Sweden for their help with RFLP typing.
Mr Iqbal Azam from Community Health Sciences, AKU, for statistical support
Mahnaz Tanveer, my colleague and friend, for helping me with the isolation and
culturing of Mycobacterium tuberculosis and supporting me through out without which I
could not have completed my work.
All my colleagues at Clinical Microbiology Lab for providing me the samples and
support in getting things started.
All my PhD and Juma Research Lab colleagues with whom I had great scientific
exchanges and shared my ups and downs of thesis progress.
Jack Fernandes and other staff members of Research department, for their support
through out my stay in the PhD program.
Dr Yasmin Amarsi and all my colleagues at School of Nursing, especially Science
faculty for their great support and forbearance during this thesis period.
University Research Council, AKU for funding this research.
Last but not the least, my dear family; my mother, late father, sisters and brothers for
their love, support and patience from the very beginning until now!
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DECLARATION
I declare this thesis does not incorporate without acknowledgement any material previously submitted for a degree or diploma in any university and that to the best of my knowledge it does not contain any material previously published or written by another person except where due reference have been made in the text.
______________________________ (Signature of candidate)
December, 2008 Date
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List of Tables
No. Title Page #
1.1: Median prevalence of MDR-TB amongst new and previously treated 27 cases by region (%) 1.2: MTB genes associated with resistance to antituberculous agents 28 2.1: Geographical Distribution of selected MTB strains for RFLP 40 2.2: Overview of number of IS6110 element present in 78 MTB Isolates 50 3.1: Geographical Distribution of selected MTB strains for MIRU typing 57 3.2: MIRU-VNTR primers for twelve loci 61 3.3: Allelic diversity of 367 MTB isolates 64 3.4: Twelve MIRU loci analysis of CAS1 and ‘unique’ spoligotypes 65 3.5: MIRU loci analysis of MDR M tuberculosis 66 4.1: PCR primers for drug resistance gene amplification 82 4.2: Detection of Rifampicin Resistance mutations by rpoB gene sequencing 83 of MDR-MTB strains 4.3: Detection of Isoniazid Resistance mutations by katG and inhA gene 84 Sequencing of MDR-MTB strains 5.1: Primers and FRET probes used for PCR amplification and detection 97 of RMP and INH resistance in MTB strains 5.2: Detection of prevalent Rifampicin and Isoniazid resistance by FRET 102 probes using real time PCR 5.3: Summary of mutations identified by Sequencing and Real time PCR in 104 Rifampicin and Isoniazid resistant MTB isolates as compared to phenotypic drug susceptibility testing (DST) 5.4: Comparison of results obtained by LiPA and DNA Sequencing on 106 33 MDR-TB strains
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List of Figures
No. Title Page # 1.1: TB incidence rate in four provinces of Pakistan 8 1.2: Tuberculosis infection 13 1.3: MTB genome showing polymorphic repeat sequences 16 2.1: Schematic diagram of steps of IS6110-RFLP method 43
2.2: Autoradiograph of Internal Marker and IS6110 probe hybridization 46
2.3: Dendrogram of IS6110-RFLP typing of Mycobacterium tuberculosis 49
3.1: Schematic diagram of steps of MIRU-VNTR method 62
3.2: Dendrogram of MIRU-VNTR typing of Mycobacterium tuberculosis 66
3.3: Composite dendrogram of RFLP and MIRU-VNTR of 78 MTB strains 67 5.1: Mechanism of action of FRET probes 96 5.2: Melting curves detecting rpoB mutation using FRET probes 101 5.3: Melting curves detecting katG mutation using FRET probes 103 5.4: Melting curves detecting inhA mutation using FRET probes 103 5.5: rpoB mutations identified by InnoLiPA assay 105
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Table of Contents
Page Abstract…………………………………………………………………………… iv List of abbreviations ……………………………………………………………… vii Acknowledgement ………………………………………………………………… viii Declaration ………………………………………………………………………… x List of tables ………………………………………………………………………. xi List of figures……………………………………………………………………….. xii Chapter One: Mycobacterium tuberculosis: An overview
1.1: General Background 1 1.2: History of tuberculosis 2 1.3: Global impact of tuberculosis 4 1.4: Pakistan 7 1.5: Tuberculosis in Pakistan 9 1.6: Natural history of M. tuberculosis 11 1.7: Genome and genomic diversity of M. tuberculosis 12 1.8: Molecular Epidemiology of Tuberculosis & its significance 14 1.9: Molecular epidemiological data from Pakistan 20 1.10: Antituberculous therapy 22 1.11: Drug-resistant tuberculosis 24 1.12: Multiple Drug resistant tuberculosis 25 1.13: MDR-TB in Pakistan 29 1.14: Molecular mechanisms of Drug resistance 29 1.15: Drug susceptibility testing 30 1.16: Goal of present study 32
Chapter Two: Genotyping of Mycobacterium tuberculosis using IS6110-Restriction Fragment Length Polymorphism
2.1: Background 35 2.2: Objectives 38 2.3: Methods
2.3.1: Mycobacterial strains 39 2.3.2: Culture and antibiotic susceptibility 39 2.3.3: DNA extraction 41 2.3.4: IS6110- RFLP typing 42 2.3.5: Computer-assisted phylogenetic analysis 47 2.3.6: Statistical analysis 47
2.4: Results 2.4.1: Diversity of RFLP 48
2.4.2: IS6110-copy number 48 2.5: Discussion 51
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Chapter Three: Genotyping of Mycobacterium tuberculosis using Mycobacterial Interspersed Repetitive Unit typing method 3.1: Background 53 3.2: Objectives 55 3.3: Methods
3.3.1: Mycobacterial strains 56 3.3.2: Culture and antibiotic susceptibility 58 3.3.3: DNA extraction 58 3.3.4: MIRU typing method 58 3.3.5: Phylogenetic analysis 60 3.3.6: Statistical analysis 60
3.4: Results 3.4.1: MIRU typing for CAS1 and ‘unique’ strains 68 3.4.2: Allelic diversity 68 3.4.3: Discriminatory power of MIRU typing for CAS1 70
3.4.4: Comparison of MIRU and RFLP typing profiles 70 3.4.5: Analysis of MIRU typing of MDR isolates 71
3.5: Discussion 72 Chapter Four: Detection of drug resistance gene mutations in MDR CAS1 and Beijing strains by Sequencing 4.1: Background 76
4.2: Objectives 78 4.3: Methods
4.3.1: Mycobacterial strains 79 4.3.2: Culture and antibiotic susceptibility 79 4.3.3: DNA extraction 79 4.3.4: Sequencing of rpoB gene for RMP resistance 80 4.3.5: Sequencing of katG and inhA genes for INH resistance 80 4.3.6: Statistical analysis 81
4.4: Results 4.3.1: rpoB gene mutations for RMP resistance 85 4.3.2: katG and inhA genes mutations for INH resistance 86
4.5: Discussion 87
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Chapter Five: Molecular methods for rapid detection of Rifamcin and Isoniazid resistance amongst the MDR strains of predominant genogroups of Mycobacterium tuberculosis 5.1: Background 90 5.2: Objectives 93 5.3: Methods
5.3.1: Mycobacterial strains, Culture & DNA extraction 94 5.3.2: RMP and INH resistance detection using FRET probe 94
based Real Time PCR 5.3.3: RMP resistance detection using InnoLiPA assay 98 5.3.4: Statistical analysis 99
5.4: Results 5.4.1: rpoB gene mutations for RMP resistance using 100
FRET probes 5.4.2: katG and inhA genes mutations for INH resistance 105
using FRET probes 5.4.3: rpoB gene mutations for RMP resistance using InnoLiPA 105
5.5: Discussion 108
Chapter Six: General discussion and Conclusions 110
References 117 Appendices 130 A: Publications 1: “Characterization of Mycobacterium tuberculosis Central Asian Strain1
using Mycobacterial Interspersed Repetitive Unit genotyping” paper published in BMC Microbiology
2: “M. tuberculosis Central Asian Strain 1 MDR isolates have more mutations in rpoB and katG genes as compared with other genotypes” paper published in Scand. Journ. of Inf. Diseases 3: “Molecular Epidemiology of Mycobacterium tuberculosis: Review article” Published in Infectious Diseases Journal, Pakistan 4: “Genotyping and Drug resistance patterns of M. tuberculosis strains in Pakistan” paper published in BMC Microbiology
B: Curriculum vitae
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Chapter One
Introduction
Mycobacterium tuberculosis: An overview
“If the importance of a disease for mankind is measured by the number of fatalities it causes, then tuberculosis must be considered much more important than those most feared infectious diseases, plague, cholera and the like. One in seven of all human beings dies from tuberculosis. If one only considers the productive middle-age groups, tuberculosis carries away one-third, and often more.”
Robert Koch, March 24, 1882
1.1: General background
More than 125 years after the discovery of its causative agent, tuberculosis (TB) is still a
major killer disease worldwide, with > 8 million new cases and >2 million deaths each
year. It is estimated that today one third of the world population is infected with TB
Chapter preview Page # 1.1: General Background 1 1.2: History of tuberculosis 2 1.3: Global impact of tuberculosis 4 1.4: Pakistan 7 1.5: Tuberculosis in Pakistan 9 1.6: Natural history of M. tuberculosis 11 1.7: Genome and genomic diversity of M. tuberculosis 12 1.8: Molecular Epidemiology of Tuberculosis & its significance 14 1.9: Molecular epidemiological data from Pakistan 20 1.10 Antituberculous therapy 22 1.11: Drug-resistant tuberculosis 24 1.12: Multi-Drug resistant tuberculosis 25 1.13: MDR-TB in Pakistan 29 1.14: Molecular basis of Drug resistance 29 1.15: Drug susceptibility testing 30 1.16: Goals of present study 32
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(WHO 2008). With the introduction of antituberculosis therapy (ATT) in1950s and the
use of Bacille Calmette Guerin (BCG) vaccine, many experts calculated that TB would
be eradicated in few years. However, TB generally and multidrug resistant (MDR;
defined as resistant to at least rifampicin (RMP) and isoniazid (INH) TB especially is
increasing worldwide since 1990. It is estimated that MDR strains of Mycobacterium
tuberculosis (MTB) constitutes 1-3% of global TB isolates (WHO 2008). World Health
Organization (WHO) estimates that up to 50 million people worldwide may be infected
with drug resistant strains of TB. The fatality rate of MDR-TB is 20-80%. In addition
human immunodeficiency virus (HIV) epidemic has also played a key role in increasing
the number of new TB cases (Aaron, Saadoun et al. 2004; WHO 2008).
Thus global increase in TB, particularly of drug resistant strains emphasize the
need for rapid detection and drug susceptibility testing of MTB in clinical samples. Such
rapid detection is necessary for adequate antituberculous therapy and containment of
resistant strains. In addition, new drugs as well as a better vaccine are also needed for
improved therapy and preventive measures.
1.2: History of Tuberculosis
TB is an ancient disease that has been called King’s Evil, lupus vulgaris,
consumption and white plague during the last several centuries. Archeological findings
from Europe, Egypt, Greece and Rome show evidence of TB (Mathema, Kurepina et al.
2006). For several centuries, scientists and physicians have been trying to understand the
nature of tuberculosis for better diagnoses, prevention, and cure. Hippocrates thought the
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disease was inherited, while Aristotle (4th century B.C.) pointed out its contagious nature.
This view of TB reemerged in the second half of 17th century, when Italian physicians
supported the idea that TB was contagious. In contrast, doctors in Northern countries,
such as Denmark and Sweden, favored hereditary causes of this disease. They believed
that the theory of TB being contagious was not proven scientifically and did not explain
why some people in urban settings did not get TB even where there was a high incidence
of the disease (Haas 1996, Little, Brown and Co. Boston, Mass). To solve this
philosophic difference, in 1865, Jean-Antoine Villemin, a French military physician,
reported transmitting TB to laboratory rabbits by inoculating tuberculous tissue from a
cadaver. This report was strongly opposed by other French medical practitioners, who
argued that there had to be more modern and more social solutions to the problem of TB,
which arose in the poorer classes due to malnutrition, poor sanitation, and overwork. The
report by Robert Koch 17 years later in 1882, which conclusively showed that TB was
indeed caused by a bacterium discredited this argument (Koch 1982). However, belief in
the societal causes of TB continued in early 20th century and they used TB as an example
of a disease that was caused by overwork and malnutrition (Barnes 2000). Finally this
dichotomy in explaining the etiology of tuberculosis was resolved in early 20th century
by Edward Trudeau's work. He showed that TB could be induced in rabbits with a
purified MTB culture but that the environmental conditions in which the animals were
maintained greatly influenced the course of the disease. This simple experiment gave
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scientific validity to the treatment of TB in sanitarium for fresh air and healthy food,
started by European physicians in the mid-1800s (Trudeau 1887).
Thus, it was concluded by Rene Dubos that TB is caused by a bacterium, but
environmental factors play a major role, and purely medical solutions alone would not
work to cure and prevent TB (Dubos 1952). This has been clearly seen by the rising
number of TB patients in the last half of the 20th century.
1.3: Global impact of Tuberculosis:
WHO estimates that one third of the world’s population is infected with MTB. In
1993, WHO declared TB a global emergency. TB accounts for 2.5% of the global burden
of disease. Although more cases of TB are reported among men, it is still the commonest
cause of death in young women (Dye, Scheele et al. 1999; Borgdorff, Nagelkerke et al.
2000). The incidence of TB ranges from less than 10 per 100,000 in North America to
100 to 300 per 100,000 in Asia and Western Russia to over 300 per 100,000 in Southern
and Central Africa (WHO 2008).
The TB incidence declined steadily during 1900s in the developed countries in
Europe and United States mainly due to improved socioeconomic conditions, BCG
vaccination and effective antituberculous therapy. This downward trend however, started
increasing in the mid-1980s. Only by extensive efforts mainly through supervised
5
antibiotic therapy has the situation been reversed in Europe and the United States
(Frieden, Fujiwara et al. 1995).
The developing world is however, still suffering from a rising incidence of TB.
Majority of TB cases in the developing world are a result of lack of health care facilities
(Waaler 2002; WHO 2008). According to WHO statistics, the African region has the
highest estimated incidence rate (356/100,000) of TB but the majority of TB patients live
in Asia where a rapid increase in population and HIV cases are the main factors
contributing to the rising number of TB cases in these areas. India, China, Bangladesh,
Indonesia and Pakistan together account for 50% of the new TB cases that arise every
year (Dye 2006; WHO 2008). The other major implication of TB is the occurrence of
disease in 75% of people within the economically productive age group i.e.15-54 years.
Since 95% of all TB cases occur in developing countries, it has a large impact on
socioeconomic development of these countries (Dye 2006; WHO 2008).
WHO has identified 22 highly endemic countries, which contribute about 80% to
the global TB burden. These countries are labeled as being high burden countries (HBCs)
of TB. These HBCs and WHO are making collaborative efforts to achieve the global
target of TB case detection (70%) and cure (85%) through Directly Observed Treatment
Short-course (DOTS) (WHO 2003). To date, however only the Philippines and Vietnam
have succeeded in meeting their targets of case detection and treatment success by the
end of 2004 (Dye 2006; WHO 2008).
6
Pakistan and its three neighboring countries India, China and Afghanistan
contribute significantly to the global TB burden. India ranks first on the list of 22 HBCs
in terms of total number of TB patients present. It is estimated that approximately 40% of
people living in Indian subcontinent, China and the Pacific Rim are infected with TB
(Dye, Scheele et al. 1999).
India with its population of over 1000 million is estimated to account for nearly
30% of the global tuberculosis burden. In 1997, the estimated incidence of TB in India
was 187 per 100,000 populations, while the current estimate is 505 per 100,000 (Dye
2006). The situation has been adversely affected by the increasing number of HIV
infected people. The HIV rate in India has been found to vary between 0.4% and 28.8%
(Chadha 2005). Overall about 50-70% of HIV positive patients in India reportedly
develop TB during their lifetime thus leading towards increasing number of TB-HIV co-
infection. It has also been estimated that TB-HIV co-infection in India has increased 2-10
times as compared to the estimates made in 1997 (Swaminathan, Ramachandran et al.
2000; WHO 2008).
China ranks 2nd on the list of 22 HBCs. It has the second largest population of
tuberculosis patients in the world, with more than 1.3 million new cases of tuberculosis
every year. It accounts for 17% of the total global burden of TB (WHO 2008).
Afghanistan is the last country on the WHO list of 22 HBCs. TB is a major public health
burden with 95,000 new TB cases and 26,000 deaths every year in the country (WHO
2008).
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1.4: Pakistan
Pakistan covers an area of 310,000 square miles in South Asia. It has four major
provinces (Punjab, Sind, North-West Frontier Province and Baluchistan) (Figure 1.1).
The most populous province, Punjab, has only 26 percent of the land area but is home to
about one-half (56% in the 1998 census) of the 143 million people estimated to live in
Pakistan. This population makes Pakistan the seventh most populous country in the world.
Life expectancy is estimated at 66 years in women and 64 years in men (Pakistani
Federal Bureau of Statistics www.statpak.gov.pk/depts/fbs/statistics/pds).
8
Figure 1.1: TB incidence rate in four provinces of Pakistan
(Adapted from pakistan.spe.org/.../clear_pakistan_map2.gif and 2007 data from NTP)
43/100,000
148/100,000
6/100,000
123/100,000
9
1.5: Tuberculosis in Pakistan
Pakistan ranks eight amongst the 22 high burden TB disease countries with an
estimated incidence rate of 181/100,000 while sputum positive TB cases are estimated at
82/100,000 population (WHO 2008). In addition there are estimated 268,000 new cases
and 64,000 deaths from TB each year in Pakistan which contributes about 44% of TB
cases in WHO Eastern Mediterranean region (WHO 2008).The quoted TB burden in
Pakistan is likely to be an underestimated figure as many cases in the country go
unreported due to lack of access to health care facility, over crowding, poverty and other
social constraints (WHO 2003). According to National Tuberculosis Control Program
(NTP) of the country, TB incidence in each of four provinces ranges between 96/100,000
in Baluchistan to 148/100,000 in Punjab (Figure 1.1).
Similar to other developing countries, majority of TB cases occur predominantly
in the economically most productive, 15 to 54-year age group, which further hinders
socioeconomic development. TB is, therefore, not only an important problem in the field
of public health in the country; it is also a socio-economic issue that not only harms
people’s health, but also imposes a heavy economic burden on the family of TB patients
(Dye 2006; Habib and Baig 2006).
In order to control the high incidence rate of TB the government of Pakistan
endorsed DOTS in 1994 in collaboration with WHO. DOTS strategy is being
implemented in Pakistan under NTP. Although treatment success by DOTS program in
Pakistan is reported at more than 70%, DOTS detection rate is below the population
10
coverage of 24%, far below the 70 percent target, suggesting that many patients do not
have access to DOTS (Netto, Dye et al. 1999; WHO 2003). In 2001, the government
declared TB a national emergency, which led to a TB budget increase from US$1.65
million in 2001 to US$26 million in 2006. This was mainly for the development in DOTS
expansion and increases in the case detection and cure rate.
Poor TB patient compliance to treatment has further exacerbated the situation.
Partially treated TB patients are the main source for the spread of drug resistant TB
particularly MDR-TB. Non-availability of drugs, lack of accessibility to the health care
centers and lack of awareness regarding consequences of incomplete therapy are known
causes of poor compliance with TB treatment (Israr 2003; Khan and Malik 2003;
Hussain, Mirza et al. 2005; Habib and Baig 2006).
High treatment cost of drug-resistant TB especially MDR-TB is an additional
economic burden both to the individual patient and society. WHO estimates that 3.4% of
all new cases have MDR-TB while in previously treated patients it is estimated at 36%
(WHO 2008).
Although HIV prevalence is low in Pakistan, HIV/TB co-infection has been
reported as 19% in high risk group (Memon, Memon et al. 2007). The high risk group
mostly include injection drug users (IDU) (Memon, Memon et al. 2007; Vermund and
Yamamoto 2007). Among female sex workers 1-2% HIV prevalence has been reported
(Ministry of Health Pakistan, 2005; National AIDS Control Program Pakistan,
2005;(WHO 2003).
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1.6: Natural history of Mycobacterium tuberculosis
Mycobacterium tuberculosis (MTB), which causes TB, is one of the most
successful pathogens of mankind and may have killed more people than any other
pathogen (Daniel 2006). These organisms are called “the Koch’s bacillus” after Robert
Koch who first identified tubercle bacilli (Sakula 1983).
MTB is one of the members of Mycobacterium tuberculosis complex (MTC).
Other species in the complex are Mycobacterium africanum, Mycobacterium bovis,
Mycobacterium bovis BCG, and Mycobacterium microti. These are slow growing,
fastidious, aerobic and acid fast rods. On solid culture medium; Lowenstein Jensen (LJ)
and 7H10/7H11 Middlebrook agar, MTB requires 16-18 hours to undergo one cycle of
replication and within 2-3 weeks a single bacterium may produce a visible colony
(Wayne 1977).
Transmission of MTB occurs mainly through air by droplets containing bacteria,
which are formed when infected people cough or sneeze. Inhalation of these small
droplets may cause pulmonary TB amongst healthy people. MTB stay in its human host
in the form of a dormant infection. MTB organisms multiply inside macrophages and
infection may persist in the latent phase for years. Reactivation of latent disease may
occur after several years of initial infection in small proportion of infected people. The
major risk factors for reactivation include old age, malnutrition, HIV co-infection or other
chronic diseases such as diabetes. However, the majority of infected people never
develop clinical disease (Figure 1.2). Prominent symptoms presented by patients are
12
chronic and productive cough, low grade fever, night sweats, fatigue and weight loss.
Extra-pulmonary TB may involve lymph nodes, kidney, bone, joints or meninges etc.
1.7: Genome and genomic diversity of Mycobacterium tuberculosis:
At the end of the 20th century complete genome sequencing of Mycobacterium
tuberculosis H37Rv revealed that it consists of 4.4 mega base pairs with densely packed
coding regions. It is estimated to include 4000 protein coding regions and has a very high
(65%) guanine and cytosine content (Cole, Brosch et al. 1998).
Comparative genome sequencing of several MTB strains such as Mycobacterium
bovis, Mycobacterium tuberculosis H37Rv and Mycobacterium tuberculosis H37Ra, have
revealed that Mycobacterium tuberculosis (MTB), the main causative agent of TB, while
displaying diverse phenotypic characteristics and host ranges, is genetically fairly
consistent with an overall genomic similarity of 99.9% throughout the world
(Boddinghaus, Rogall et al. 1990; Sreevatsan, Pan et al. 1997). Studies have shown that
MTB with highly conserved genome also has polymorphic genomic regions called
repetitive sequences. MTB genome is characterized by the presence of numerous
repeated sequences whereas no plasmid is detected in this species. Genetic diversity
among MTB strains in a particular geographical location has been linked with these
polymorphic repeat sequences (Sreevatsan, Pan et al. 1997). These repeat sequences,
which include insertion sequence (IS), direct repeat (DR) and tandem repeats, are widely
used for MTB strain typing in molecular epidemiology of tuberculosis.
13
Figure 1.2: Tuberculosis infection
(Adapted from www.nature.com/.../v5/n8/full/nri1666.html)
14
Based on these polymorphic repeat sequences various molecular techniques have
been introduced. Molecular epidemiological studies using these molecular techniques
have helped identifying predominant MTB genotype in a defined geographical location.
1.8: Molecular epidemiology of tuberculosis and its significance
Molecular epidemiology is an integration of molecular biology with epidemiology.
Recent developments in molecular biology have resulted in techniques that allow rapid
identification and tracking of specific strains of M. tuberculosis as they spread through
the population (Barnes and Cave 2003; Filliol, Driscoll et al. 2003; Garcia de Viedma
2003; Filliol, Motiwala et al. 2006). While, previous methods, such as colony
morphology, comparative growth rates, susceptibility to antibiotics, and phage typing
were useful but did not provide sufficient information regarding TB epidemiology. In the
early 1990s, different molecular methods were described to discriminate between MTB
isolates (van Embden, Cave et al. 1993; van Soolingen, de Haas et al. 1993). Most of
these techniques use DNA polymorphism based on repetitive DNA elements of M.
tuberculosis as genetic markers. Each of these methods results in strain specific genetic
profiles (fingerprints). Identical strain fingerprints are called clusters, which are usually
associated with recent transmission. While strains with unique fingerprints represent
remote transmission or infection acquired in past.
Several molecular epidemiological studies of tuberculosis have been carried out
using various polymorphic repeat sequences i.e. IS, DR and tandem repeats (Figure 1.3).
IS elements are small DNA segment that can be inserted at multiple sites. These elements
15
show high level of genetic polymorphism and widely used for studying the genetic
variability in MTB species. IS6110-RFLP typing method, based on IS6110 copy numbers
and positions, has been used worldwide as the gold standard TB epidemiology because of
reproducibility of the results (van Embden, Cave et al. 1993). However, there are certain
limitations of this technique. Firstly, around 20% of MTB isolates contains few or no
copies of IS6110 element and the method is unreliable for typing such strains. Secondly,
it needs around 4.5µg of DNA which takes several weeks to culture enough viable
organisms. In addition, the method is labor intensive, technically demanding and
expensive (van Soolingen et al, 1995).
Various PCR based techniques, which target polymorphic loci other than IS6110,
have also been used in epidemiological studies of TB. Spacer oligonucleotide typing
(spoligotyping) based on the polymorphism in DR locus is a widely used molecular
typing method for epidemiological studies worldwide (Kamerbeek, Schouls et al. 1997).
The 36 bp size DRs are interspersed by unique spacer DNA sequences of 35-41 bp.
Spoligotyping identifies the presence or absence of 43 spacer DNA sequences between
the variable direct repeats using PCR in a particular MTB strain. Spoligotyping is simple,
rapid and highly reproducible. However, Spoligotyping has less discriminatory power as
it targets less than 0.1% MTB genomic area as compared to IS6110 based typing which
examines the entire genome (Figure 1.3). Therefore, it cannot totally replace IS6110-
RFLP typing because of its lower discriminatory power, except for in strains with low
copy numbers of IS6110 (van Soolingen et al, 2001).
16
Figure 1.3: MTB genome showing polymorphic repeat sequences
Hypothetical genome of MTB strain X and polymorphic repetitive sequences such as
IS6110, DR and MIRUs for genotyping
(Reference: Adapted from Barnes, P et al, 2003)
Mycobacterial Interspersed Repetitive Units (MIRU)
Direct Repeats (DR)
Insertion Sequence 6110 (IS6110)
17
VNTR-MIRU is another PCR based genotyping method which has higher
discriminatory power than spoligotyping. MIRU-VNTR is based on detection of
independent mini satellite like loci scattered through out the MTB genome and has been
shown to be a reliable and reproducible typing method for studying the MTB population
structure in different geographical locations (Dale, Nor et al. 1999; Supply, Lesjean et al.
2001; Cowan, Mosher et al. 2002; Sola, Filliol et al. 2003). The typed strains are
expressed by a 12-digit numerical code, corresponding to the number of repeats at each
locus (Supply, Mazars et al. 2000; Mazars, Lesjean et al. 2001). This numerical code is
easy to compare and exchange at inter-, and intra-laboratory level. The discriminatory
power of MIRU-VNTR analysis is proportional to the number of loci evaluated. Twelve
loci based MIRU-VNTR analysis has been used in a number of molecular epidemiologic
studies and to elucidate the phylogenetic relationship of clinical isolates (Sola, Filliol et al.
2003; Supply, Warren et al. 2003; Sun, Lee et al. 2004; Warren, Victor et al. 2004;
Kremer, Au et al. 2005). The discriminatory power of standard twelve loci based typing
only slightly lower than that of the IS6110-RFLP, which is currently the gold standard for
MTB genotyping (Supply, Lesjean et al. 2001). However MIRU-VNTR has several
advantages over gold standard IS6110-RFLP method; it requires little culture growth for
DNA and provides comparable, numerical data by using standard gel electrophoresis or
by automated analysis using fluorescence tagged PCR primers and sequencer. MIRU
typing is also a method of choice for MTB strains with 0-5 copies of IS6110 element, as
have been reported from south Asian countries (Mazars, Lesjean et al. 2001).
18
Using these molecular tools molecular epidemiological studies have identified
several MTB strains so far including Beijing family (van Soolingen, Qian et al. 1995),
Haarlem family (Kremer, van Soolingen et al. 1999), Delhi (Bhanu, van Soolingen et al.
2002), the Cameroon family (Niobe-Eyangoh, Kuaban et al. 2004), the Latin American
Mediterranean(LAM) family, the East African Indian (EAI) clade (Sola, Filliol et al.
2001; Filliol, Driscoll et al. 2002; Filliol, Driscoll et al. 2003; Brudey, Driscoll et al.
2006) etc.
Molecular epidemiological studies have also greatly facilitated identification of
TB transmission locally as well as globally. They have helped investigate whether
particular clinical strains of TB differ in infectivity, severity of disease or drug
susceptibility and to differentiate between recent and or previous infection.
Molecular epidemiological investigations to study transmission dynamics by
exploring genetic diversity of MTB strains are of prime importance. These help to
evaluate the patterns and dynamics of TB transmission in a defined geographical location.
When this technique is applied similar MTB strains can be identified, which can lead to
important clues about the pattern and dynamics of transmission. Two of the earliest such
studies conducted in the United States, one in San Francisco and one in New York City,
investigated MTB isolates with matching DNA fingerprints with the assumption that they
were epidemiologically related and represented recent transmission of TB among the
patients (Small, Hopewell et al. 1994). This finding led the authors to conclude that as
many as 40% of TB cases in these two cities were the result of recent transmission and
that TB control practices in San Francisco and New York were not effectively decreasing
19
MTB transmission. Thus these data were used to implement effective interventions to
stop such outbreaks.
Molecular epidemiological studies have also assisted in unraveling the differences
in virulence and drug susceptibility related to bacterial genetic background (Silver, Li et
al. 1998; Valway 1998; Caminero 2001; F.Barnes 2003). Such as Beijing strain (W
strain) have shown higher growth rate in human macrophages as compared to other MTB
isolates (Zhang M 1999). In addition Beijing strains have also found to have a greater
association with drug resistance and have also been found to be less protective against
BCG vaccination in infection as compared to other MTB isolates (H.McShane 2003). A
clinical strain CDC1551 has been shown to induce a stronger immune response in
experimental animals as compared the other isolates (Menca C 1999; Zhang M 1999).
Molecular epidemiological studies help determining genotype specific drug
resistance mutations and prevalence of these mutations in a population (Mathema,
Kurepina et al. 2006). Studies have been conducted to describe clonal expansion of drug
resistant MTB strains between patients in a defined geographical location (Bifani 2001,
Bifani1996, Post FA 2004). Such studies help understanding the relationship between
genetic background, drug resistance and transmission of TB in a population and might
assist in taking measures to prevent the spread of resistant TB strains.
20
Among its applications, molecular epidemiology has also served to elucidate the
poorly understood role of relapses and exogenous reinfection of persons with recurrent
TB after cure (Small, Shafer et al. 1993). In addition the technology has also been applied
as a complementary tool to conventional methods in TB laboratory to investigate and
control cross-contamination.
Thus, to summarize, molecular epidemiology has greatly helped in the analysis of
disease transmission and its prevention. This information plays a pivotal role in
investigating the relationship of a specific genotype with phenotypic characters such as
disease causation and development of antimicrobial resistance.
1.9: Molecular epidemiological data from Pakistan
There is increasing evidence that the genetic difference of MTB is strongly
associated with specific geographical locations (Yang, Barnes et al. 1998; Sola, Devallois
et al. 1999; Soini, Pan et al. 2000; Mazars, Lesjean et al. 2001; Filliol, Driscoll et al.
2003; Hirsh, Tsolaki et al. 2004; Mokrousov, Narvskaya et al. 2004; Gutierrez, Ahmed et
al. 2006). Thus molecular epidemiological studies in Pakistan, a high TB incidence
country may provide unique insights into dissemination dynamics and virulence of the
pathogen. However limited molecular typing data is available from the country. Although
limited, this molecular data shows the predominance of some specific strain types in
Pakistan. A study based on samples collected from Rawalpindi revealed 37% of 113
MTB isolates investigated showed common profile based on five loci based VNTR (A to
21
E exact tandem repeat) analysis. On the other hand a study from Peshawar using IS6110
based typing reported that 89% (n=8/9 ) of isolates were of a common strain type (Sechi,
Zanetti et al. 1996; Gascoyne-Binzi, Barlow et al. 2002). Limitation of these studies is
small sample size and use of only one genotyping technique. In addition the strain types
identified in these studies were not matched with any global database.
Recently spoligotyping was used to investigate strain types amongst 314 MTB
isolates from the country. After comparing the results with international database
(SpolDB3) CAS1 strains lacking spacers 4-7 and 23-34 were found to be the most
prevalent (39%) in Pakistan (Hasan, Tanveer et al. 2006). The CAS1 strain has also been
reported as the second most predominant group in South Asia; India (16-22%) and
Bangladesh (17%) (Bhanu, van Soolingen et al. 2002; Banu, Gordon et al. 2004; Deepak
2005; Hasan, Tanveer et al. 2006). Although Beijing strains, lacking spacers 1-34, are the
most widely reported genotype world wide (Agerton T. B 1999; Bifani 1999; Glynn,
Whiteley et al. 2002; Li, Whalen et al. 2002; Mokrousov, Narvskaya et al. 2002; Reed,
Domenech et al. 2004), their prevalence amongst MTB isolates from Pakistan was found
to be only 6% (Hasan, Tanveer et al. 2006).
Despite the predominance of CAS1 in South Asia, there is limited data related to
its transmission dynamics, disease process and drug resistance. MIRU-VNTR typing
method, as discussed earlier, with higher discriminatory power has been used in several
studies to elucidate intra-strain genetic differences and phylogenetic relationship of
clinical isolates (Sola, Filliol et al. 2003; Supply, Warren et al. 2003; Sun, Lee et al.
2004; Warren, Victor et al. 2004; Kremer, Au et al. 2005). Twelve loci based MIRU-
22
VNTR analysis has been used in a number of molecular epidemiologic studies. It has also
been used to study Beijing strains from East and South Asia (Dale, Nor et al. 1999; Anh,
Borgdorff et al. 2000; Chan, Borgdorff et al. 2001; Prodinger, Bunyaratvej et al. 2001;
Mokrousov, Narvskaya et al. 2004; Chin, Chiu et al. 2007). Available data for MIRU-
VNTR typing for MTB in Pakistan is limited to one report wherein five exact tandem
repeat (ETR) were used to type 113 MTB isolates from Rawalpindi, Pakistan. This
showed clustering of one third of the isolates (Gascoyne-Binzi, Barlow et al. 2002).
However pertaining to CAS1, the predominant strain type in Pakistan no MIRU-VNTR
data is available.
1.10: Antituberculous therapy
Until middle of the 20th century there was no definitive treatment available for TB.
Streptomycin (S) was made available for the TB treatment. Then other drugs including
isoniazid (INH), rifampicin (RMP), pyrazinamide (PYZ) and ethambutol (E) were also
used for antituberculous therapy (ATT). Standard ATT for pulmonary tuberculosis
comprises a six month regimen. For the first two months patients receive a combination
of three to four of these drugs i.e. isoniazid, rifampicin, pyrazinamide and in some cases
ethambutol. Final four months patients continue with isoniazid and rifampicin. This six
months therapy is called “short-course” anti-tuberculous therapy (Blomberg, Spinaci et al.
2001; Moulding, Le et al. 2004). But longer courses of up to two years may be needed
for persons having TB with resistant strains or have TB-HIV co- infection and makes it
100 times as expensive as the first line regimen.
23
In early 1990s World Health Organization introduced directly observed treatment
short course (DOTS) strategy as a cost effective way to control TB (Pfyffer, Welscher et
al. 1997; Sawert, Kongsin et al. 1997; Dye, Garnett et al. 1998). DOTS strategy includes
case detection, standard short-course therapy (with three drugs out of five), regular drug
supply and monitoring and evaluating the program. DOTS has been regarded as the
highly successful and most cost effective intervention for the treatment of drug
susceptible TB control (Burman, Dalton et al. 1997; Chaulk, Friedman et al. 2000;
Chaulk and Grady 2000). However despite of high success rate, DOTS has been found
insufficient for the management of MDR-TB (Becerra, Freeman et al. 2000; Espinal, Kim
et al. 2000). Thus DOTS-Plus has been introduced which takes into account specific
issues such as drug susceptibility and careful use of second-line antituberculous drugs in
order to combat global threat of MDR-TB (Farmer and Kim 1998). In addition
implementation of DOTS-Plus has been shown extremely important to contain super
resistant extensively drug resistant (XDR) MTB strains (Kam and Yip 2004; Laserson,
Thorpe et al. 2005; Caminero 2006). XDR-TB is the disease caused by bacteria that are
resistant to any fluoroquinolones and resistant to at least one second line injectable drug
(amikacin, capreomycin or kanamycin) (Dukes Hamilton, Sterling et al. 2007). The
global emergence of XDR-TB strains poses an additional threat to the success of
tuberculosis therapy. XDR-TB has been reported from throughout the world with highest
known rates in Eastern Europe and Asia (Dukes Hamilton, Sterling et al. 2007). Since
24
XDR-TB are resistant to all the drugs considered essential in the treatment of MDR-TB
cases, the XDR-TB might be almost incurable (Gandhi, Moll et al. 2006).
1.11: Drug-resistant tuberculosis
The resistance of MTB strains to anti-tuberculosis drugs was noted when
streptomycin (S) was first used as monotherapy for TB in the 1940s. In the subsequent
years with the addition of RMP, PYZ and E, multiple antituberculous therapy was
implemented to combat emergence of single drug resistance amongst MTB strains
(Mitchison 1985; Iseman and Madsen 1989).
Drug resistance is divided into two types: primary and secondary (or acquired)
resistance. Primary resistance is defined as resistance in persons who have not received
anti-tuberculosis drugs for more than 1 month. These patients are presumed to be initially
infected with drug-resistant strains. Acquired resistance is defined as resistance to anti-
tuberculosis drugs, which arises during treatment due to poor compliance or improper
management. Adult patients can be infected with primary drug-resistant strain or acquire
resistance to anti-tuberculosis drugs during the treatment. Usually, children have primary
resistance, as they get infected from adult source with drug-resistant TB (Guidelines
1998; Pablos-Mendez, Raviglione et al. 1998).
WHO and International Union against Tuberculosis and Lung Diseases
(IUATLD) recommends using the terms drug resistance among new cases and drug
25
resistance among previously treated cases. Drug resistance among new cases (formerly:
primary drug resistance) is the presence of drug-resistant strain of M. tuberculosis in a
newly diagnosed patient who has never received anti-tuberculosis drugs or has received
them for less than 1 month. Drug resistance among previously treated cases is that found
in a patient who has previously received at least 1 month therapy with anti-tuberculosis
drugs(WHO 2003).
1.12: Multi-Drug resistant tuberculosis (MDR-TB)
Multiple-drug resistant tuberculosis (MDR-TB) is defined as simultaneous in-
vitro resistance of MTB strains to at least RMP and INH (with or without resistance to
other drugs). As RMP and INH are the most potent first line antituberculous drugs, the
emergence of MDR-TB can give rise to potentially untreatable form of the disease.
MDR-TB treatment require use of second-line drugs (SLDs) that are less effective, more
toxic and more costly than the first line based treatment (Cole 1994; Cole and Telenti
1995). In addition mortality is significantly higher among persons infected with MDR
strain than of those infected with sensitive strain. Patients with multi-drug resistant TB
remain infectious for longer time increasing the risk of disease transmission
(Drobniewski and Wilson 1998; Rattan, Kalia et al. 1998).
Despite of implementation of multiple antituberculous therapies, a steady increase
in the frequency of TB with single and multiple drug resistant MTB strains has been
reported through out the world (Bloch, Cauthen et al. 1994). In early 1990s outbreaks of
26
MDR-TB received global attention (Edlin, Tokars et al. 1992). Nosocomial outbreaks of
MDR-TB have been reported in the USA, France and other countries (CDC 1991; CDC
1991).
A recent WHO survey for the period of 2002-2006 report median prevalence of
MDR as high as 22.3% amongst new tuberculosis cases in Kazakhstan, in contrast to
14.2% in an earlier report (Ang, Ong et al. 2008). The survey also found alarmingly high
rates in other countries such as Ukraine (16% percent), Russia (15 %), and Uzbekistan
(14.8%). Other high MDR-TB areas include Estonia (12%), Lithuania and Latvia (9%),
Russian Federation and China (10%). In Iran and India MDR-TB has been reported at 5%
and 3.4% respectively (WHO 2008). India and China the two most populous countries
are major concerns for MDR-TB as these two countries account for 40% of all TB cases
worldwide. Global occurrence of MDR-TB is about 3% (Espinal, Laszlo et al. 2001;
Sharma and Mohan 2004; Jou, Chen et al. 2005), ranging from 0% in various states of
America to 22.3% in Kazakhstan (Espinal, Laszlo et al. 2001; WHO 2008). Prevalence
of MDR-TB among new and previously treated TB cases is 1.1% and 7% respectively
(Dukes Hamilton, Sterling et al. 2007). 75% of these MDR-TB cases occur in Asia
(Espinal, Laszlo et al. 2001). The global prevalence of MDR-TB amongst new and
previously treated cases has been summarized in Table 1.1.
27
Table 1.1: Median prevalence of MDR-TB amongst new and previously treated cases by region (%)
Region
MDR-TB amongst
new cases
MDR-TB amongst
previously treated cases
Africa 1.4 5.9
America 1.1 7.0
Eastern Mediterranean 0.4 48.3
Europe 0.9 4.7
South East Asia 1.3 20.4
Western pacific 0.9 15.5
Overall median 1.1 7.0
(Adapted from WHO report 2008)
28
Table: 1.2: MTB genes associated with resistance to antituberculous agents
Antituberculous
agents
Gene
Size (bp)
Product
Mutation frequency
among resistant MTB isolates (%)
Rifampicin rpoB 3,534 subunit of RNA polymerase
> 95
Isoniazid katG oxyR-ahpC
inhA
kasA
2,205 585 810
1,251
Catalase-peroxidase Alkylhydroreductase
Enoyl-ACP reductase
-Ketoacyl- ACP reductase
60-70 20 <10
<10
Streptomycin rpsL rrs
372 1,464
Ribosomal proteinS12 16s rRNA
60 <10
Ethambutol embCAB 1,164 Arabinosyltransferases
70
Pyrazinamide pncA 560 Amidase 70-100 Ethionamide inhA
ethA
810
1467
Enoyl-ACP reductase
Flavoprotein monooxygenase
<10
NA*
Kanamycin rrs 1,464 16s rRNA 65 Fluoroquinolone gyrA
gyrB
2,517
2,142
DNA gyrase subunit
DNA gyrase subunit
>90
NA
*data not available
(Adapted from (Musser 1995; Mathema, Kurepina et al. 2006)
29
1.13: MDR-TB in Pakistan
The high incidence of tuberculosis in Pakistan is also compounded by the
increasing emergence of drug resistant strains including MDR. Community based data
from country reports 1.8% prevalence of MDR-TB amongst untreated patients while
laboratory based data reports 4% of MDR-TB in untreated patients (Javaid, Hasan et al.
2008; Rao, Irfan et al. 2008). However WHO estimates 3.4% and 36% MDR-TB in new
TB patients and in previously treated patients respectively, as mentioned earlier. The
reasons for high rate of drug resistance include improper prescription, non-compliance
and over the counter sale of anti-TB drugs (WHO 2008).
Laboratory based studies from urban Rawalpindi showed an increasing frequency
of MDR from 14% in 1999 to 28% in 2004 (Butt, Ahmad et al. 2004) while study from a
tertiary care center in Karachi documented 47% MDR-TB prevalence (Irfan, Hassan et al.
2006).
1.14: Molecular basis of drug resistance
The mechanisms of drug resistance are chromosomal, caused by accumulation of
one or more mutations in independent genes. Accumulation of a number of drug
resistance mutations result in multiple drug resistance (Sreevatsan, Pan et al. 1997). Such
as resistance to RMP is well characterized and more than 95% of the RMP resistant have
mutations in an 81bp hot spot region (codon 507-533), or Rif Resistance Determining
30
Region (RRDR) of the 3534bp rpoB gene (Telenti, Imboden et al. 1993; Musser 1995).
This is the strongest correlation between phenotypic and genotypic resistance in MTB
discovered so far. This rpoB gene encodes the β subunit of RNA polymerase. RMP
interferes with the transcription and elongation of the RNA by binding to the DNA
dependent RNA polymerase (Telenti, Imboden et al. 1993; Telenti 1997).
In contrast to RMP, INH resistance is controlled by a more complex genetic
system, involving several genes such as katG, inhA, kasA, oxyR and ahpC (Zhang, Heym
et al. 1992; Banerjee, Dubnau et al. 1994; Kelley, Rouse et al. 1997). Although the
frequency of mutation at these loci varies between different population, studies show that
70-80% of INH resistance is mostly associated with mutation in codon 315 of katG and
inhA genes (Zhang, Heym et al. 1992; Banerjee, Dubnau et al. 1994). Similarly S, PYZ, E
and Fluoroquinolones resistance have been linked with mutations in rrs, pncA, embB and
gyrA genes respectively (Table 1.2).
1.15: Drug susceptibility testing
Detection of drug resistance is performed by culturing MTB in the presence of
antibiotics. These methods have been performed on egg-based or agar-based solid media
directly or indirectly. For the direct method, media are inoculated with decontaminated
and concentrated clinical specimen, while for indirect method; media are inoculated with
a bacterial suspension of the isolated strains. Based on solid media, there are three
31
conventional phenotypic methods for drug susceptibility testing: the proportion method,
the resistance ratio method and the absolute concentration method (Canetti, Fox et al.
1969). More recent methods are based on liquid media including the BACTEC
radiometric and the Mycobacterial Growth Indicator Tube methods (MGIT) (Pfyffer,
Welscher et al. 1997). However, due to the long time period necessary to obtain results
and laboriousness of these methods molecular approaches have been proposed
Molecular methods are based on the genetic determinants of drug resistance rather
than phenotypic resistance. These including DNA sequencing, real time PCR,
microarrays and kit based line probe assays has enabled detection of drug resistance in
MTB from several weeks to a few days (Torres 2002; Hillemann, Rusch-Gerdes et al.
2007). These methods include amplification of targeted gene segment correlating with
drug resistance by polymerase chain reaction (PCR), sequencing, solid-phase
hybridization, real-time PCR assay or microarray technique (Garcia de Viedma 2003).
Molecular methods have a pivotal role in identifying prevalent drug resistance
mutations amongst MTB population in a particular geographical location. Molecular
tools and genetic determinants of drug resistance has made timely diagnosis of MTB drug
resistance and adequate antituberculous therapy possible (Torres 2002; Hillemann,
Rusch-Gerdes et al. 2007). Although studies have shown both Central Asian strain1
(CAS1) and Beijing family strains as the predominant genogroups (39% and 6%
respectively) in Pakistan and in other South Asian countries there is limited data available
32
pertaining to type and frequency of drug resistance gene mutations amongst these
predominant genogroups (Bhanu, van Soolingen et al. 2002; Glynn, Whiteley et al. 2002;
Banu, Gordon et al. 2004; Almeida, Rodrigues et al. 2005; Hasan, Tanveer et al. 2006). It
is important to understand and study the drug resistance mutations in CAS1 and Beijing,
strains to develop tests for rapid detection of resistance among TB strains from Pakistan.
1.16: Goals of present study
Molecular epidemiological studies, based on the assumption that patients infected
with clustered strains are epidemiologically linked, have helped understanding the
transmission dynamics of disease. Appropriate genotyping tools are a prerequisite for
performing molecular epidemiological studies of MTB strains. Although, these tools are
well established in developed countries, establishment and use of only spoligotyping
technique has been shown in Pakistan (Hasan, Tanveer et al. 2006). However, given the
fact that spoligotyping is limited in its ability to discriminate, additional molecular
methods are required to be established for further analysis of genetic diversity of
predominant MTB genogroups in the country.
Hasan et al have shown significant association of Beijing genotype MTB strains
with MDR (Hasan, Tanveer et al. 2006). Although the most prevalent CAS1 strains have
not shown significant association with drug resistance, given the burden of CAS1 they
constitute almost fifty percent of the total MDR strains in the country.
33
While mutation in MDR Beijing strain is characterized in studies from different
geographical locations (Suresh, Singh et al. 2006; Lipin, Stepanshina et al. 2007), there is
no data on drug resistance genes mutations amongst CAS1 strains. Knowledge of type
and frequency of mutations amongst MDR strains of prevalent genogroup within a
defined geographical location is essential for the development of tools for early diagnosis
and control of MDR-TB strains (Ahmad, Araj et al. 2000; Lipin, Stepanshina et al. 2007),
no such data is available from the country. Thus there is a need to investigate mutations
among MDR CAS1 and Beijing strains from the country.
Therefore the hypotheses for this study were:
1. The prevalent MTB genogroup in the Country has intra strain
genetic diversity.
2. The prevalent drug resistant MTB strains have some common
mutations in the drug resistance genes
Thus the overall aim of this study was to explore genetic diversity amongst the
predominant genogroup of Mycobacterium tuberculosis from the country as well as to
investigate genetic basis of drug resistance amongst these predominant MTB strains.
Specific goals of this study were:
1. To contribute to the development and establishment of additional
molecular typing tools for MTB
2. To study genetic differences amongst identified prevalent genogroup,
CAS1.
34
3. To investigate the genetic basis of drug resistant (MDR) MTB strains
prevalent in our community (CAS1 and Beijing).
Information from this study would help contribute towards an understanding of
molecular epidemiology of MTB in our community. It would enable an analysis and
understanding of strain types prevalent locally. In particular it would greatly
contribute to analyzing factors leading to drug resistance at a molecular level. This
knowledge would assist to take appropriate measures for the prevention and
adequate treatment of the tuberculosis disease including MDR.
35
Chapter Two
Genotyping of Mycobacterium tuberculosis using IS6110- Restriction Fragment Length Polymorphism
2.1: Background
Insertion sequences (IS) are small mobile genetic elements which are widely
distributed throughout the MTB genome. Over 14 different kinds of IS have been
identified in the MTB genome which are usually less than 2.5 kb in size (van Soolingen,
de Haas et al. 2000). IS generate genetic polymorphism and therefore are used to
discriminate between different MTB strains. The most widely utilized IS in
epidemiological studies is IS6110. In 1990 Thierry identified IS6110 element in MTB
Chapter preview Page # 2.1: Background 35 2.2: Objectives 38 2.3: Methods
2.3.1: Mycobacterial strains 39 2.3.2: Culture and antibiotic susceptibility 39 2.3.3: DNA extraction 41 2.3.4: IS6110- RFLP typing 42 2.3.5: Computer-assisted phylogenetic analysis 47 2.3.6: Statistical analysis 47
2.4: Results 2.2.1: Diversity of RFLP 48
2.2.2: IS6110-copy number 48 2.5: Discussions 51
36
strains (Thierry, Cave et al. 1990). IS6110 is 1,355 bp in size and has 28 bp inverted
repeats at its ends. It is randomly distributed throughout the MTB genome with copy
numbers ranging between 0-26 (Kurepina, Sreevatsan et al. 1998; McHugh and Gillespie
1998). In 1993, van Embden and colleagues proposed a standardized Southern blot
hybridization method based on the frequency of IS6110 in the MTB genome which
allows strain differentiation.
Restriction fragment length polymorphism (RFLP) using insertion sequence
IS6110 was recommended by van Embden and colleagues for MTB genotyping (van
Embden, Cave et al. 1993). The method is based on the detection of differences in the
numbers and locations of the insertion element IS6110 within the strains of MTB
chromosomes.
Despite certain limitations, such as the need for large amounts of DNA, labor-
intensivity and its low discriminatory ability in cases of strains with few copy of IS6110
element (please see chapter one, section 1.8), IS6110-RFLP typing has played a
significant role in understanding the transmission patterns, source, and spread of MTB
strains (Gopaul, Brown et al. 2006; Mathema, Kurepina et al. 2006). Limited IS6110-
RFLP typing data for MTB strains from Pakistan is available (Sechi, Zanetti et al. 1996;
Dale, Al-Ghusein et al. 2003). The study reports IS6110-RFLP typing of nine MTB
isolates from Peshawar. The results showed that 89% of isolates tested had a common
strain type.
37
Following a preliminary spoligotyping report that described CAS1 strains to be
the most prevalent (39%) genogroup in Pakistan (Hasan, Tanveer et al. 2006), the present
investigation was focused on characterizing CAS1 strains using IS6110-RFLP typing
method. Although spoligotyping is a simple and rapid genotyping technique for MTB, it
is less informative for determining genetic diversity of predominant strains in the high
endemic TB areas like Pakistan (Mathema, Kurepina et al. 2006). IS6110-RFLP is a gold
standard genotyping technique for MTB, with high discriminatory power, and ability to
reveal genetic diversity within predominant strains, such as CAS1, identified on the basis
of spoligotyping.
38
2.2: Objectives
The specific objectives of this study were:
2.2.1: To establish IS6110-RFLP genotyping technique 2.2.2: To identify IS6110-RFLP profile to further characterize and analyze selected CAS1 strains in comparison with ‘orphan’ types strains
2.2.3: To identify utility of IS6110 typing in MTB isolates from Pakistan
2.2.4: To use Bio-numeric software program for the fingerprint analysis
.
39
2.3: Methods
2.3.1: Mycobacterial strains
926 previously spoligotyped MTB strains which were collected between
2003-2005 from across the country were stored within our strain bank (Hasan,
Tanveer et al. 2006; Tanveer, Hasan et al. 2008). 78 isolates (43 CAS1 and 35
orphan types) were randomly selected from 314 spoligotyped strains (Table 2.1)
for characterization with IS6110-RFLP (Hasan, Tanveer et al. 2006). During the
optimization phase of establishment of RFLP, results from MTB DNA grown on
Lowenstein Johnson (LJ) medium was compared with results from MTB DNA
grown on Middlebrook medium. In our experience MTB DNA from LJ gave
better RFLP results. In this study, therefore we used DNA from strains grown on
LJ medium.
M. tuberculosis reference strain 14323 (obtain on request from van
Embden, National Institute of Public Health and the Environment, the
Netherlands) was used as external control and SDL-PvuII marker as internal
control.
2.3.2: Culture and antibiotic susceptibility
All 78 selected strains were cultured on LJ. Drug susceptibility data for all
78 strains was collected from the Clinical Laboratory of Aga Khan University
Hospital, where phenotypic drug resistance testing was performed using the
standard agar proportion method on enriched Middlebrook 7H10 medium (Wayne
and Krasnow 1966; Standards. 1995; Isenberg 2004).
40
Table 2.1: Geographical Distribution of selected MTB strains for RFLP
Location
CAS1 (n: 43)
Unique (n: 35)
Total (n: 78)
Karachi 11 10 21
Sind (other than Karachi)
12 10 22
Punjab 12 11 23
NWFP 7 3 10
Baluchistan 1 1 2
(Reference: (Hasan, Tanveer et al. 2006))
41
The following final drug concentrations were used: rifampicin, 1µg/ml
and 5µg/ml; isoniazid, 0.2µg/ml and 1µg/ml; streptomycin, 2µg/ml and 10µg/ml;
ethambutol 5µg/ml and 10µg/ml. Pyrazinamide was tested using BACTEC (7H12
medium, pH 6.0, at 100µg/ml, Becton Dickinson) as per manufacturer’s
instructions. Strains with a high level of resistance for rifampicin (5µg/ml) and
isoniazid (1µg/ml) were further selected for MDR analysis.
2.3.3: DNA extraction
Genomic DNA was extracted from the MTB strains cultured on LJ and 7H10
agar medium, via cetyl-trimethylamonium bromide (CTAB) method after heat
inactivating the samples at 85 oC for 45 minutes (Honore-Bouakline, Vincensini
et al. 2003). 50 μl lysozyme (10 mg/ml) was added and incubated at 37 oC for
over night. After that 60 μl of 10% SDS and 15 μl Proteinase K (10 mg/ml) was
added and vortexed for few seconds and incubated at 65 oC in a water bath for an
hour. Then 100 μl of CTAB of and100 μl 5M NaCl was added and vortexed. till
all the samples became milky. Then it was incubated at 65 oC for an hour. 750 μl
of Chloroform and Isoamyl (24:1) was added, vortexed for 30 second and then
centrifuged at 14000 rpm at room temperature for 15 min for phase separation.
The aqueous phase was separated in another autoclaved eppendorf and 450 μl
isopropanol was added to precipitate the nucleic acid. The samples were placed at
-20o C for over night. Next day samples were centrifuged at 14000 rpm for 15
minutes. Then supernatant was decanted without loosing the DNA pellet. 1 ml of
42
70% ice cold Ethanol was added and centrifuged at 12000 g for 5 minutes.
Supernatant was discarded and pellet was dried and then reconstituted in 50 -
100μl of 1X TAE. Optical density of DNA was taken and maintained in the
record.
2.3.4: IS6110- RFLP typing method (Standard van Embden method)
The established protocol, used by National Institute of Public
Health and the Environment, the Netherlands, (van Embden, Cave et al.
1993) was followed to type seventy-eight clinical MTB strains DNA,
extracted from MTB culture grown on LJ, was digested with PvuII and
Southern blots were hybridized with IS6110 probe. Blots were normalized
with the standard M. tuberculosis reference strain 14323 (figure 2.1). Brief
outline of the method has been given below.
IS6110 probe synthesis
IS6110-probe (based on 245 bp region of digested IS6110 element)
was prepared by PCR technique, using DNA template of BCG strain (P3)
of Mycobacterium tuberculosis (which was received on request from
National Institute of Public Health and the Environment the Netherlands)
with 50-ng/l of the following primers:
INS-1 (5’ CGT GAG GGC ATC GAG GTG GC)
INS-2 ( 5’GCG TAG GCG TCG GTG ACA AA)
43
Figure 2.1: Schematic diagram of steps of IS6110-RFLP method
(Adapted from Daley, 2005)
44
For PCR one cycle of 3 minutes at 95C, 40 cycles consisting;
1minute at 95C and 1 minute at 62C, 1 cycle of 2 minutes at 72C and
finally 10 minutes extension at 72C. PCR product was then run on 1%
agarose gel to check the size and purity of amplified product.
PCR product was purified using spin column Qiagen PCR Product
Purification kit as per manufacturer’s instructions. The purified IS6110
probe was then labelledwith Horseradish Peroxidase using ECL
(Enhanced Chemiluminesence, Amersham) labeling kit as per the
instructions provided by the manufacture.
PvuII restriction digestion of the sample DNA
Restriction digestion was carried out for 2 - 4.5 g of DNA sample
of each of our strain as well as control strains P3 and MTB 14323 with
PvuII enzyme. The digested DNA was run on a small 0.8% agarose gel in
order to confirm the digestion.
Gel electrophoresis
The digested DNA of each sample as well as control MTB 14323
strains were run on 20 x20 cm size, 0.8% agarose gel on 20V over night
with SDL-PvuII Internal Marker (which was prepared using Supercoiled
DNA ladder, digested with PvuII enzyme, and PhiX174 as per standard
protocol).
45
The gel was then visualized using UV transilluminator to check the
migration of the band of the external marker Mt14323. then the gel was
placed on an UV transilluminator (Biometrica Whatman Co., Model
1003464) until the fluorescence of the ethidium bromide fades completely.
Southern blotting
The blots from the gel were transferred on nitrocellulose
membrane using vacuum blotter.
Probe hybridization
The blots on nitrocellulose membrane were hybridized with
Enhaced Chemiluminescent (ECL) labeled IS6110 probe as well as with
Internal Marker probe over night in the rolling bottle placed in
hybridization chamber at 42C.
Blot development
Blots on the membrane for IS6110 and Internal Marker were
developed on the X-ray film using Amersham ECL detection system
(Figure 2.2).
46
2.2-A
2.2-B
Figure 2.2: Autoradiograph after the hybridization of MTB strains with Internal Marker probe (A) and IS6110 probe (B)
47
2.3.5: Computer-assisted phylogenetic analysis of fingerprints
The autoradiographs of IS6110-RFLP were scanned using Scanjet 3400-
hp scanner. Bionumerics Software (Applied Maths) was used to analyze the
molecular patterns generated by IS6110-RFLP. This experiment was repeated at
least in duplicates for all 78 strains. Scanned images of IS6110 blots were
uploaded in Bionumerics program. Then the dendrogram was generated by
unweighted pair group method analysis (UPGMA). Strains were classified in a
cluster when they shared hundred percent identical IS6110-RFLP patterns.
2.3.6: Statistical analysis
Association of number of IS6110 element and strain type was determined
by chi-square using version 16 of SPSS (Special Program for Social Sciences
Software, USA). A P value of < 0.05 was considered significant.
48
2.4: Results
2.4.1: Diversity of RFLP
IS6110-RFLP typing of 78 strains (43 CAS1 and 35 ‘unique’ spoligotyped
strains) resulted in 73 different RFLP types (Figure 2.2). One cluster of two
unique strains, with single copy of IS6110 was identified; the remaining seventy-
two strains revealed unique RFLP patterns.
2.4.2: IS6110-copy number
The copy number of IS6110 in each of the isolates was estimated as
determination of exact copy number was difficult in some of the strains due to
band intensity. Of 78 strains, 4 (5%) strains had ‘zero’ copy, 6 (7.7%) had low
(1-5) copy, 9(11.5%) had medium (6-10) copy and 59(75.6%) had high (11-17)
copy of IS6110 element. Two out of four zero copy strains belonged to CAS1
family while all the low copy isolates belonged to unique strain type (Table 2.2).
Occurrence of low copy IS6110 element (0-5) was significantly associated (P <
0.05) with unique strains.
Of 68 high copy IS6110 strains (>5 copies), 60% belonged to CAS1 strain
type. There was an average of 12.8 IS6110 copies in CAS1 strain, and 9.2 in
unique strains. While CAS1 strains were not clustered a 60% homology was
observed amongst their IS6110 profile. In addition there was no similarity
between IS6110 profiles of MDR strains.
49
Figure 2.3: Dendrogram of IS6110-RFLP typing of Mycobacterium tuberculosis
The figure illustrates analysis of IS6110-RFLP of 78 MTB strains using Bionumerics
software (Applied Maths). The strains included 43 CAS1 and 35 ‘unique’ exhibiting
heterogeneous IS6110-RFLP profiles with one cluster of two strains with one copy
IS6110 while four strains are IS6110 deficient. * Denotes CAS1 strains exhibiting 60%
homology despite of heterogeneous IS6110-RFLP profiles.
* *
*
*
*
4 Zero copy strains
50
Table 2.2: Overview of number of IS6110 element present in 78 MTB Isolates
# of
IS6110 element
# of Strains TOTAL
PercentageCAS1 (43) Unique (35)
MDR S* MDR S
0
2
-
-
2
4
5 %
1
-
-
2
3
5
6.4 %
2-5
-
-
1
-
1
1.2 %
6-10
2
-
2
5
9
12 %
11-15
7
24
2
18
51
65.3 %
16-17
3
5
-
-
8
10 %
14
29
7
28
78
* S = Susceptible MTB strain 0-5 band = 10/78 (13%) 11-17 band = 59/78 (76%)
51
2.5: Discussions
IS6110-RFLP analysis revealed diverse profiles of MTB strains isolated in
Pakistan. RFLP analysis of our strains showed only one cluster of two MTB strains
belonging to unique strain type while none of the CAS1 strains were clustered. This
finding from this study is in agreement with previous studies, which have shown an
association between MTB strain diversity and high TB incidence. (Sahadevan, Narayanan
et al. 1995; Vukovic, Rusch-Gerdes et al. 2003). Strain diversity has also been reported
from neighboring countries; India, Iran and Bangladesh (Farnia, Mohammadi et al. 2004;
Storla, Rahim et al. 2006; Chauhan, Sharma et al. 2007).
Overall 87% of MTB strains showed occurrence of more than six copies and 13%
of MTB strains had 0-5 copies of IS6110 element tested in this study. 41/43 CAS1strains
contained more than six IS6110 copies. This data is comparable with data reported in
other studies from subcontinent (Das, Narayanan et al. 2005; Chauhan, Sharma et al.
2007; Mathuria, Sharma et al. 2008). Two zero copy IS6110 element CAS1strains have
been reported first time in this study (which were ensured by repeating the RFLP
experiment at least thrice for these strains). The high genetic polymorphism in the region
may be due to enhanced genetic variation and transposition of IS6110 element, which
results in variable profiles (Sreevatsan 1997; Brosch 2002).
Overall this study shows that IS6110-RFLP could effectively be used for the
molecular epidemiological studies to characterize majority of MTB isolates from the
country. However despite its use IS6110-RFLP has some practical limitations; in addition
52
to the need of 4.5µg of DNA, the technique is expensive. It is moreover difficult to
characterize large number of MTB isolates using RFLP due to the fact that RFLP testing
is labour intensive. Finally comparison of RFLP base strain information with global data
bases requires expensive and sophisticated computer analysis which is not widely
available (Mathema, Kurepina et al. 2006). In view of these shortcomings an additional
simple method with comparable discriminatory power with IS6110-RFLP is required
which can be used to characterize MTB isolates in general from the country. The next
objective of the study therefore was the characterization of MTB isolates using a second
typing method i.e MIRU-VNTR.
53
Chapter Three
Genotyping of Mycobacterium tuberculosis using Mycobacterial Interspersed Repetitive Unit typing method
3.1: Background
VNTR-MIRU typing method has widely been used for genotyping of clinical
MTB strains. The typed strains are expressed by a 12-digit numerical code,
corresponding to the number of repeats at each locus (Supply, Mazars et al. 2000; Mazars,
Lesjean et al. 2001). This numerical code is easy to compare and exchange at inter-, and
Chapter preview Page # 3.1: Background 53 3.2: Objectives 55 3.3: Methods
3.3.1: Mycobacterial strains 56 3.3.2: Culture and antibiotic susceptibility 58 3.3.3: DNA extraction 58 3.3.4: MIRU typing method 58 3.3.5: Phylogenetic analysis 60 3.3.6: Statistical analysis 60
3.4: Results 3.4.1: MIRU typing for CAS1 and ‘unique’ strains 68 3.4.2: Allelic diversity 68 3.4.3: Discriminatory power of MIRU typing for CAS1 70
3.4.4: Comparison of MIRU and RFLP typing profiles 70 3.4.5: Analysis of MIRU typing of MDR isolates 71
3.5: Discussions 72
54
intra-laboratory level. Therefore a number of studies have used standard twelve loci
based MIRU-VNTR typing method to elucidate the phylogenetic relationship of clinical
isolates in molecular epidemiologic studies (Sola, Filliol et al. 2003; Supply, Warren et
al. 2003; Sun, Lee et al. 2004; Warren, Victor et al. 2004; Kremer, Au et al. 2005).
In this study we have used standard twelve loci based MIRU-VNTR typing
method to characterize CAS1 and ‘orphan’ spoligotyped MTB strains selected from
different geographical locations in Pakistan as described by Supply (Supply, Lesjean et al.
2001).
55
3.2: Objectives
The specific objectives of this part of the study were:
3.2.1: To establish VNTR-MIRU typing method in lab for MTB strains
3.2.2: To identify VNTR-MIRU profile of CAS1 and ‘orphan’ spoligotyped
strains selected from different geographical locations in Pakistan
3.2.3: To identify the most discriminatory MIRU loci for CAS1 as compared
with ‘orphan’ spoligotyped strains
3.2.4: Determine the association of MIRU loci with MDR of clinical isolates
3.2.5: Compare the MIRU-VNTR analysis of selected MTB strains with their
IS6110-RFLP profile.
56
3.3: Methods
3.3.1: Mycobacterial strains
A total of 926 strains of Mycobacterium tuberculosis were spoligotyped in
a parallel study at the Aga Khan University (Hasan, Tanveer et al. 2006; Tanveer,
Hasan et al. 2008). These MTB isolates were collected during the period of 2003–
2005 from the four provinces of Pakistan by a stratified random sampling method
(Hasan, Tanveer et al. 2006). These strains were stored at the MTB strain bank of
the Aga Khan University Hospital. From these stored strains a total of 178 CAS1
strains and 189 ‘unique’ isolates were selected (Table 3.1) for this study using
following statistical formula:
Formula: n = Npq (N-1)D + pq
Where,
N = total no. of isolates available
P = probability (as 40% of CAS1so 0.40 & 60% of Unique so 0.60)
q = probability of failure (1-p)
D = (bound on error) .05 = B2 = (.05)2 = .0006 4 4
57
Table 3.1: Geographical Distribution of selected MTB strains for MIRU typing
Location
CAS1 (n: 178)
Unique (n: 189)
Total (n: 367)
Karachi 49 80 129
Sind (other than Karachi)
33 34 67
Punjab 78 46 124
NWFP 16 25 41
Baluchistan 2 4 6
(Reference: Hasan 2006 & Tanveer 2008)
58
3.3.2: Culture and antibiotic susceptibility
Selected CAS1 and unique MTB strains were taken from the MTB strain
Bank of the Aga Khan University Hospital. Revival and culturing of the strains
were carried out in the Biosafety Level III, Juma Research laboratory of the Aga
Khan University. All mycobacterial strains were cultured on Middlebrook 7H10
agar. Susceptibility testing was performed by the standard agar proportion method
in the Clinical Laboratory of the Aga Khan University Hospital as discussed in
detail in Chapter Two. Strains with a high level of resistance for rifampicin
(5µg/ml) and isoniazid (1µg/ml) were further selected for MDR analysis.
3.3.3: DNA extraction
Genomic DNA was extracted from the MTB strains cultured on 7H10 agar via
cetyl-trimethylamonium bromide (CTAB) method similarly as described in
Chapter Two, Section 2.3.3.
3.3.4: MIRU-VNTR typing
MIRU-VNTR PCR (As described by Supply, 2001)
After extraction of DNA, PCR was performed for twelve MIRU loci (2, 4,
10, 16, 20, 23, 24, 26, 27, 31, 39 and 40) individually for all 367 isolates using
specific primers (Table 2.1) as described previously (Supply, Lesjean et al. 2001).
59
Each of the PCR master mixes contained 0.4µM concentration of specific primers,
0.5mM concentration of dNTPs mix, 1mM concentration of MgCl2, 1x PCR
buffer, 4 % of DMSO and 1U of Super Tth Taq DNA polymerase for a 25 µl
reaction. Master mixes were distributed to 96-well plates. Approximately 40-60ng
of template DNA was added for each sample. M tuberculosis H37Rv DNA used
as a positive control while negative controls lacked DNA. PCR plates were sealed
and placed in PerkinElmer 9700 thermo cycler starting with a denaturing step of
15 min at 95oC, followed by 35 cycles of 1 min at 94oC, 1 min at 59oC, and 1 min
30 s at 72oC, followed by an extension of 72oC. After the thermo cycling step, all
367 MTB isolates were analyzed using a simple gel electrophoresis method. The
MIRU-VNTR method has been outlined in Figure 2.1.
Allele scoring of MIRU loci
The PCR products were electrophoresed on a 2.5% agarose gel and sized
with a 100-bp ladder (Promega). Band sizes were measured using Geldoc
Quantity-one (Bio-RAD) soft ware and allelic numbers were determined using the
MIRU-VNTR allele scoring table from international database link
www.ibl.fr/mirus.html.
60
3.3.5: Phylogenetic analysis
The twelve digits MIRU-VNTR allele score obtained for each MTB strain
was then entered into Bionumerics soft ware (Applied Maths, St. Martens
Latem, Belgium) as a character set and used to generate a dendrogram by un-
weighted pair group using arithmetic averages (UPGMA). To compare isolates
combining both methods, a multi experiment composite data set with MIRU and
Spoligotyping was created by using the available tools in Bionumerics.
3.3.6: Statistical analysis
The Hunter Gaston Discriminatory Index (HGDI) was calculated for
comparison of discriminatory power of MIRU-VNTR typing for different loci
(Hunter and Gaston 1988).Non parametric analysis was carried out using the
Mann-Whitney test to determine the utility of MIRU typing to distinguish
between CAS1 and ‘unique’ as well as CAS1-MDR and ‘unique’ MDR. A P
value of < 0.05 was considered significant. This analysis was carried out using
version 14 of SPSS (Special Program for Social Sciences Software, USA).
61
Table 3.2: MIRU-VNTR primers for twelve loci
Loci # PRIMER sequences (5' - 3')
MIRU 2 2F - TGGACTTGCAGCAATGGACCAACT
2R - TACTCGGACGCCGGCTCAAAAT
MIRU 4
4F - GCGCGAGAGCCCGAACTGC
4R - GCGCAGCAGAAACGTCAGC
MIRU 10
10F - GTTCTTGACCAACTGCAGTCGTCC
10R - GCCACCTTGGTGATCAGCTACCT
MIRU 16
16F - TCGGTGATCGGGTCCAGTCCAAGTA
16R - CCCGTCGTGCAGCCCTGGTAC
MIRU 20
20F - TCGGAGAGATGCCCTTCGAGTTAG
20R - GGAGACCGCGACCAGGTACTTGTA
MIRU 23
23F - CTGTCGATGGCCGCAACAAAACG
23R - AGCTCAACGGGTTCGCCCTTTTGTC
MIRU 24
24R - CGACCAAGATGTGCAGGAATACAT
24F - GGGCGAGTTGAGCTCACAGAA
MIRU 26
26F - TAGGTCTACCGTCGAAATCTGTGAC
26R - CATAGGCGACCAGGCGAATAG
MIRU 27
27F - TCGAAAGCCTCTGCGTGCCAGTAA
27R - GCGATGTGAGCGTGCCACTCAA
MIRU 31
31F - ACTGATTGGCTTCATACGGCTTTA
31R - GTGCCGACGTGGTCTTGAT
MIRU 39
39F - CGCATCGACAAACTGGAGCCAAAC
39R - CGGAAACGTCTACGCCCCACACAT
MIRU 40
40F - GGGTTGCTGGATGACAACGTGT
40R - GGGTGATCTCGGCGAAATCAGATA
(Reference: Supply, 2001)
62
Figure 3.1: Schematic diagram of steps of MIRU-VNTR method (*Adapted from Supply 2000 )
Amplicon size is measured using agarose gel
Based on the size of the amplicons, 12 digit code is assigned to each strain
*MIRU typing is based on 12 out of 41 Polymorphic MIRUs
PCR for each of the 12 MIRU loci Is carried out for each strain
The numerical code is then entered in Bio-numeric Software program
63
Figure 3.2: Dendrogram of MIRU-VNTR typing of Mycobacterium tuberculosis
Three hundred and sixty seven strains were typed and a cluster analysis was carried out using Bionumerics software using the unweighted pair group method. The 178 CAS1 strains studied showed an overall homology of >70%. No MIRU clusters were observed between any of the 187 ‘unique’ strains studied.
64
Table 3.3: Allelic diversity of 367 MTB isolates from Pakistan
Discriminatory Index: #≥ 0.6= Highly Discriminant and *0.3-0.59= Moderately Discriminant.
MIRU loci
Allele Number Allelic Diversity
Rank
Conclusion
0 1 2 3 4 5 6 7 8 92 18 85 262 2 0.4355 11 *Moderately
discriminant
4 21 13 290 9 11 22 1 0.3670 12 Moderately discriminant
10 18 4 29 48 73 133 50 8 4 0.7862 3 #Highly discriminant
16 43 19 46 126 83 40 9 1 0.7885 2 Highly discriminant
20 22 60 248 29 3 4 1 0.5080 10 Moderately discriminant
23 11 4 4 10 49 234 42 8 4 1 0.5616 8 Moderately discriminant
24 77 238 34 2 3 12 1 0.5271 9 Moderately discriminant
26 9 24 20 12 31 58 96 84 30 3 0.8337 1 Highly discriminant
27 20 23 107 167 42 8 0.6893 7 Highly discriminant
31 18 4 30 101 116 73 25 0.7731 4 Highly discriminant
39 22 60 153 116 15 1 0.6962 6 Highly discriminant
40 11 31 116 150 51 6 2 0.7073 5 Highly discriminant
Average 0.6394
65
Table 3.4: Twelve MIRU loci analysis of CAS1 and ‘unique’ spoligotypes.
MIRU Loci
HGDI values for P- value
CAS1 strains (n=178)
Conclusion ‘unique’ strains (n= 189)
Conclusion
2 0.4953 Moderately discriminant
0.3859 Moderately discriminant 0.105
4 0.2676 Poorly discriminant
0.4540 Moderately discriminant 0.000*
10 0.7449 Highly discriminant 0.8190 Highly
discriminant 0.004*
16 0.7503 Highly discriminant
0.7760 Highly discriminant 0.325
20 0.5993 Moderately discriminant 0.4242 Moderately
discriminant 0.124
23 0.5211 Moderately discriminant 0.6099 Highly
discriminant 0.126
24 0.5709 Moderately discriminant 0.4966 Moderately
discriminant 0.436
26 0.8117 Highly discriminant 0.8511 Highly
discriminant 0.000*
27 0.7588 Highly discriminant 0.6151 Highly
discriminant 0.909
31 0.7772 Highly discriminant 0.7756 Highly
discriminant 0.340
39 0.7090 Highly discriminant
0.6775 Highly discriminant 0.732
40 0.6970 Highly discriminant 0.7228 Highly
discriminant 0.661
Average: 0.6419 Average: 0.6339
Significantly different loci are indicated by ‘*’ (P<0.05)
66
Table 3.5: MIRU loci analysis of MDR M tuberculosis
MIRU Loci
HGDI values for P- value
MDR CAS1 (n=62)
Conclusion
MDR ‘unique’ (n= 54)
Conclusion
2 0.5944 Moderately discriminant
0.4983 Moderately discriminant 0.463
4 0.2871 Poorly discriminant
0.4689 Moderately discriminant 0.007*
10 0.7536 Highly discriminant 0.8092 Highly
discriminant 0.142
16 0.7784 Highly discriminant 0.7582 Highly
discriminant 0.188
20 0.6076 Highly discriminant
0.3662 Moderately discriminant 0.098
23 0.5463 Moderately discriminant
0.5311 Moderately discriminant 0.862
24 0.5219 Moderately discriminant 0.4780 Moderately
discriminant 0.266
26 0.8080 Highly discriminant 0.8609 Highly
discriminant 0.100
27 0.7504 Highly discriminant
0.5542 Moderately discriminant 0.955
31 0.7583 Highly discriminant
0.7659 Highly discriminant 0.934
39 0.7155 Highly discriminant 0.6003 Highly
discriminant 0.465
40 0.7150 Highly discriminant 0.7358 Highly
discriminant 0.332
Average: 0.6530
Average: 0.6189
*Locus 4 between the two is significantly different (P value < 0.05)
67
Figure 3.3: Composite dendrogram of IS6110-RFLP and MIRU-VNTR of 78 MTB strains The figure illustrates a composite analysis of IS6110-RFLP and MIRU-VNTR of 78 MTB strains using Bionumerics software (Applied Maths).
68
3.4: Results
3.4.1: MIRU typing for the predominant CAS1 genogroup and ‘unique’
strains from Pakistan
The twelve loci MIRU-VNTR analysis detected a total of 349 MIRU
patterns in our sample size of 367 strains (Figure 3.2). The 178 strains of the
CAS1 genogroup were found to be more than 70 % homologous, but were further
divided into 160 distinct patterns comprising of; 15 clusters of two strains each, 1
cluster of four strains and with 144 non-matching patterns. The 189 strains
previously identified by spoligotyping as ‘unique’ remained un-clustered after
MIRU analysis. The distribution of the MIRU alleles is summarized in Table 3.3.
3.4.2: Allelic diversity
Allelic diversity of clinical isolates was determined by twelve MIRU
loci analysis using the Hunter Gaston Discriminatory Index (HGDI). Overall,
MIRU-VNTR typing of 367 MTB strains indicated a discriminatory power of
0.999. Diversity of CAS1 (n: 178) and ‘unique’ (n: 189) strains was further
calculated separately (Table 3.3 and 3.4). Allelic analysis of 178 CAS1 strains
showed a HGDI of 0.998.
69
Allelic diversity for each locus was calculated in order to determine the
discriminatory power of these loci in a combined group for the MTB population
studied. Overall, the average allelic diversity of loci studied in these strains was
found to be 0.6394 (Table 3.3). Based on their discriminatory index (DI), seven
MIRU loci 10, 16, 26, 27, 31, 39 and 40 were designated as “highly discriminant”
(DI 0.6). While, MIRU loci 2, 4, 20, 23 and 24 were designated as “moderately
discriminant” (0.3 DI 0.6) (Sola, Filliol et al. 2003). In our MTB population
locus 26 was found to be the most discriminatory allele in order to distinguish
between CAS1 strains and ‘unique’ spoligotypes. Locus 26 provided a 10 allelic
discrimination with a HGDI of 0.833. This was followed by loci 16, 10, 31, 40,
39 and 27 respectively in order of decreasing discrimination. Locus 4 was found
to be the least discriminatory with 7 alleles and a HGDI of 0.367.
As shown in Table 3.4, the average allelic diversity of CAS1 strains was found to
be 0.6419. Of these, seven MIRU loci, numbers 26, 31, 27, 16, 10, 39, and 40,
were “highly discriminant” (DI: ≥ 0.6); four MIRU loci, 20, 24, 23, and 4 were
“moderately discriminant” (DI: 0.3-0.59); while locus number 4 was “poorly
discriminant” (DI< 0.3) for CAS1 isolates.
The average allelic diversity of ‘unique’ strains was found to be 0.6339 (Table
3.4). The diversity patterns observed for ‘unique’ strains was similar that found
70
for CAS1 strains , i.e. eight MIRU loci, number 26, 10, 16,31, 40, 39, 27 and 23
were “highly discriminant” (DI: ≥ 0.6) and four loci numbers 24, 4, 20 and 2 were
“moderately discriminant” (DI:0.3-0.59). However, no loci for ‘unique’ strains
were identified to be “poorly discriminant”.
3.4.3: Discriminatory power of MIRU typing for CAS1
Further statistical analysis was carried out to investigate the utility of each
of the twelve loci of MIRU typing to distinguish between CAS1 and ‘unique’
strains. Data was analyzed using the non-parametric Mann-Whitney test. Results
revealed that differences in loci 4, 10 and 26 were statistically significant (P-value
< .01).
3.4.4: Comparison of MIRU and RFLP typing profiles
To further investigate the heterogeneous pattern shown by MIRU-VNTR
and IS6110-RFLP typing, MIRU and RFLP profiles of 78 MTB strains were
compared which comprised a subset of strains; 43 CAS1 and 35 ‘unique’
spoligotypes. IS6110-RFLP typing of these 78 strains resulted in 73 different
RFLP types (Figure 3.3). One cluster of two strains, with single copy of IS6110
was further discriminated into individual patterns by MIRU-VNTR typing. The
remaining seventy-two strains revealed unique RFLP patterns.
71
3.4.5: Analysis of MIRU typing of MDR isolates
We analyzed MIRU patterns for all the MDR strains in order to investigate
an association between resistance and MIRUs. Of the CAS1 strains studied, 62
were MDR (35%) while 54 ‘unique’ strains were MDR (29%). HGDI values of
MIRU loci in MDR strains are shown in Table 3.5. Locus 4 was found to be
statistically significant in discriminating between CAS1 and ‘unique’ MDR
strains. Overall, no significant difference could be established between MIRU
patterns of MDR isolates belonging to either CAS1 or ‘orphan’ strains.
72
3.5: Discussion
Using 12 loci based MIRU-VNTR typing 367 MTB strains were studied and were
found to be highly diverse. Of the 178 CAS1 strains studied only 34 (19%) clustered into
groups based on MIRU profiles, while all 189 ‘unique’ spoligotypes studied had non-
matching MIRU profiles and therefore remained unclustered.
Twelve loci based MIRU-VNTR typing has been extensively used to study
Beijing strains. Results of these studies indicates Beijing strains to display variable
clustering, between 53-100% (Banu, Gordon et al. 2004; Mokrousov, Narvskaya et al.
2004; Kovalev, Kamaev et al. 2005; Kremer, Au et al. 2005; Nikolayevskyy 2006).
Amongst the Beijing isolates, locus 10 has been found to be the most discriminatory
followed by locus 26 and 31, while other loci being almost monomorphic (Mokrousov,
Narvskaya et al. 2004; Nikolayevskyy 2006). In contrast, 12 loci based MIRU-VNTR
analysis of CAS1 strains exhibited diverse MIRU-VNTR profiles, which did not reveal
any monomorphic loci within the CAS1 genogroup. Our data showed MIRU loci 26, 31,
16, 10, 27, 39 and 40, in decreasing order, to be the most discriminatory for the CAS1
genogroup of Mycobacterium tuberculosis. Despite exhibiting genetic variability CAS1
strains studied also revealed more than 70% homology in their MIRU profile. Altogether
these results are in accordance with previous findings which have suggested that the
definition of ongoing transmission in high TB incidence areas should include closely
73
related MIRU-VNTR genotypes (Yeh, Ponce de Leon et al. 1998; Hanekom, van der
Spuy et al. 2008).
Overall allelic diversity and discriminatory power of the VNTR loci in the MTB
isolates of CAS1 and ‘ unique’ spoligotypes studied in this study were higher than that
reported earlier for strains from Singapore, Russia and South Africa (Mokrousov,
Narvskaya et al. 2004; Sun, Bellamy et al. 2004). The greater diversity observed can be
attributed to continual import of new strains due to traffic of people between Pakistan and
neighboring countries endemic for tuberculosis such as, migration of populations from
Afghanistan, and also travel between neighboring countries including China, Iran, the
Middle East, India and Bangladesh.
To further understand the genetic character of MTB strains studied, we compared
a subgroup of CAS1 and ‘unique’ strains to IS6110-RFLP typing (discussed in Chapter
2). One cluster of two strains detected by RFLP typing containing one copy of IS6110
was further differentiated by MIRU-VNTR typing, further supporting the higher
discriminatory ability of MIRU-VNTR typing especially for low copy IS6110 strains
(Blackwood, Wolfe et al. 2004) .
We also compared our MIRU profiles of the CAS1 family isolates with studies
from Russia, Singapore and Bangladesh (Banu, Gordon et al. 2004; Mokrousov,
Narvskaya et al. 2004; Sun, Bellamy et al. 2004; Gutierrez, Ahmed et al. 2006), and also
74
with CAS strains from India (Gutierrez, Ahmed et al. 2006). However, none of the CAS1
MIRU types identified in this study were shared by those reported previously.
We have used the standard 12 loci based method of MIRU-VNTR typing.
However, recent studies have identified increasing numbers of related MIRU loci which
may help in further discrimination between strains. Supply et al. used 29 loci based
typing and subsequently recommended 24 loci based typing for phylogentic analysis and
15 loci typing for improved epidemiological studies (Supply, Allix et al. 2006). They
identified MIRUs 10, 26, 40, 31, 4 and 16 as being highly discriminatory (in decreasing
order) for routine epidemiological studies (Supply, Allix et al. 2006). On the other hand
Gutierrez et al used 21 loci based VNTR typing to study 91 MTB isolates from India
(Gutierrez, Ahmed et al. 2006).
Overall analysis of MIRU loci for MTB strains revealed loci 26, 16, 10, 31, 40, 39,
27, 23, 24, 20, 2 and 4 to be in descending order of discrimination for allelic diversity.
Loci 4, 10 and 26 had a significantly lower discriminatory index with a P-value <0.05 in
CAS1 strains than in ‘unique’, suggesting these loci to be the most conserved in CAS1
strains. In a region where CAS1 family of strains are the most prevalent spoligotype we
found MIRU loci 26, 31, 16, 10, 27, 39 and 40, in decreasing order, to be the most
discriminatory for differentiation of Mycobacterium tuberculosis.
75
In addition, locus 4 of CAS1 MDR strains showed significantly lower
discriminatory index with a P-value <0.05 when compared with MDR ‘unique’
spoligotype strains.
Prevalence of MDR-TB amongst untreated patients in Pakistan is reportedly 1.8%
(Javaid, Hasan et al. 2008). However, laboratory based studies have reported MDR-TB
prevalence of up to 47% in their samples (Irfan 2006; Butt 2004). CAS1 is the most
prevalent MTB genotype in the country followed by the Beijing genogroup (Hasan 2006).
The Beijing family of strains has been shown to be associated with drug resistance in
China, Russia, Vietnam, New York, and Estonia (Glynn 2002), no such association with
drug resistance has been demonstrated for CAS1 strain.
In view of increasing MDR burden detailed analysis of MDR strains is required.
Therefore we further studied the common mutations in drug resistance genes in the MDR
strains of predominant genogroups CAS1 and Beijing as well as in unique MTB strains.
76
Chapter Four
Detection of drug resistance gene mutations in MDR CAS1 and Beijing strains by Sequencing
4.1: Background
Drug resistance in MTB results from accumulation of resistance mutations within
chromosomes (Sreevatsan, Pan et al. 1997). More than 95% of the RMP resistance have
mutations in the rpoB gene. Whereas mutations in katG and inhA genes are associated
with 70-80% of INH resistance (Zhang, Heym et al. 1992; Banerjee, Dubnau et al. 1994;
Kelley, Rouse et al. 1997). Knowledge of type and frequency of mutations amongst
Chapter preview Page # 4.1: Background 76 4.2: Objectives 78 4.3: Methods
4.3.1: Mycobacterial strains 79 4.3.2: Culture and antibiotic susceptibility 79 4.3.3: DNA extraction 79 4.3.4: Sequencing of rpoB gene for RMP resistance 80 4.3.5: Sequencing of katG and inhA genes for INH resistance 80 4.3.6: Statistical analysis 81
4.4: Results 4.3.1: rpoB gene mutations for RMP resistance 86 4.3.2: katG and inhA genes mutations for INH resistance 87 4.5: Discussions 88
77
prevalent genogroup has been shown to be essential for the development of appropriate
tools for early diagnosis and control of MDR-TB strains (Lipin, Stepanshina et al. 2007).
There is limited information on mutations leading to drug resistance within MTB strains
from the country. One study reports transition at codon 531 of the rpoB gene in 7 out of
9 rifampicin resistant MTB isolates from Pakistan (Rossau, Traore et al. 1997). Studies
from India and Russia have shown an association of Beijing genogroup strains with
mutation at codon 531 of rpoB gene (Suresh, Singh et al. 2006; Lipin, Stepanshina et al.
2007). However no information regarding drug resistance gene mutations in CAS1 strains
is available so far.
Given the fact that CAS1 is the most predominant genotype in Pakistan their
association with drug resistance needs to be investigated. Therefore in this part of study
prevalent mutations in the drug resistance genes for RMP and INH resistance were
investigated in MDR strains of predominant genogroups.
78
4.2: Objectives
The specific objectives of this study were to:
4.2.1: Identify mutations in rpoB gene associated with Rifampicin resistance amongst CAS1, Beijing and ‘orphan’ Multi-drug resistant strains
4.2.2: Identify mutations in katG and inhA genes associated with Isoniazid
resistance amongst CAS1, Beijing and ‘orphan’ MDR strains
4.2.3: Analyze possible associations of specific mutation conferring Rifampicin
and Isoniazid resistance with prevalent genogroups as well as with ‘orphan’ MTB isolates.
79
4.2: Methods
4.2.1: Mycobacterial strains
All the available CAS1, Beijing and unique MDR strains, which were
genotyped previously, were included in this study. Thus a total of 62 MDR strains
comprised of 30 CAS1, 12 Beijing and 20 unique strains were selected for this
study. 10 susceptible MTB Susceptible strains including eight CAS1 and two
Beijing strains were also included. Laboratory strain Mycobacterium tuberculosis
H37Rv was used as control (wild type).
4.2.2: Culture and antibiotic susceptibility
All mycobacterial strains were cultured on Middlebrook 7H10 agar.
Susceptibility testing was performed by the standard agar proportion method as
discussed in chapter two. Strains with a high level of resistance for rifampicin
(5µg/ml) and isoniazid (1µg/ml) were further selected for MDR analysis.
4.2.3: DNA extraction
Genomic DNA was extracted from the MTB strains cultured on 7H10
agar via cetyl-trimethylamonium bromide (CTAB) method similarly as described
in Chapter Two, Section 2.3.3.
80
4.2.4: Sequencing of rpoB gene for Rifampicin resistance
rpoB gene was amplified using specific primers and cycles as reported
previously (Ma, Wang et al. 2006), using Perkin Elmer thermo cycler. 495 bp
rpoB region covering 81bp hyper variable region was amplified using specific
primers (Table 4.1). The larger rpoB region was targeted in order to explore
mutations outside hyper-variable region as well. For PCR one cycle of 15 minutes
at 95C, 35 cycles consisting; 30 seconds at 95C, 1 minute at 62C, 30 seconds
at 72C and finally 07 minutes extension at 72C was performed. Amplicons were
purified using QIA quick Qiagen PCR purification kit. Purified amplicons were
then sent for the direct sequencing of both the strands to Macrogen Company,
Korea. DNA sequences were then compared using BLAST from NCBI web link
(www.ncbi.nlm.nih.gov/BLAST).
4.2.5: Sequencing of katG and inhA genes for Isoniazid resistance
For detection of INH resistance, katG, and promoter region of inhA genes
were amplified using specific primers and cycles as reported reviously (Gonzalez,
Torres et al. 1999; Ahmad, Fares et al. 2002; Ma, Wang et al. 2006) using Perkin
Elmer thermo cycler.
428 bp katG region covering codon 315 and inhA region covering
regulatory region of inhA gene was amplified using specific primers (Table 4.1).
81
For PCR one cycle of 15 minutes at 95C, 35 cycles consisting; 30
seconds at 95C, 1 minute at 67C, 30 seconds at 72C and finally 07 minutes
extension at 72C was performed. Similarly amplicons were purified using QIA
quick Qiagen PCR purification kit and then sent for sequencing to Macrogen
Company, Korea. DNA sequences were then compared using BLAST from NCBI
web link (www.ncbi.nlm.nih.gov/BLAST).
4.2.6: Statistical analysis
Association of a particular gene mutation with a genogroup was
determined by chi-square using version 16 of SPSS (Special Program for Social
Sciences Software, USA). A P- value of < 0.05 was considered significant.
82
Table 4.1: PCR primers for drug resistance gene amplification
Antimicrobial agent
Target gene
Primers
Amplicon size (bp)
Reference
Rifampicin rpoB F: GACGACATCGACCACTTC R: GGTCAGGTACACGATCTC
495 Ma X, 2006
Isoniazid katG F: CACTGGCCGCGGCGGTCGACATT R: GTCAGTGGCCAGCATCGTCGGGGA
423 Ahmad, 2002
inhA F: CCTCGCTGCCCAGAAAGGGA R: ATCCCCCGGTTTCCTCCGGT
248 Gonzalez, 1999
83
Table 4.2: Detection of Rifampicin Resistance mutations by rpoB gene sequencing of MDR-MTB strains
# tcc(2),ccg(6),ctc(2),tac(1), cgc(1), aac(1),gac(1) *denotes significant difference (P < 0.05) between the occurrence of a specific site mutation as compared with other mutations as analyzed for each subgroup (CAS1, Beijing and Unique strains) ^ Mutations observed in susceptible strains also
Geno-type (N)
Number of Codon mutation
Hyper variable region (codon 507-533)
Outside Hyper variable region (codon 534-604) 511 gct
ccg
513 caa cta
515 atg ata
516 gac gtc
522 tcg ttg
526 cac#
527 aag cag
531 tcg ttg
539 tca gtc
541 gac tac
544 ctg ccg
548 cgc ctc
549 gac tac
550 gtg ttg
561 Atc gtc
573 gaa gca
^592 ggg gag
^595 tac acc
None
CAS1 (30)
1 - - 1 2 13* 1 17 2 - - 1 1 1 - - 4 3 1
Beijing (12)
- - - 2 - - - 8 - 1 - - - 1 1 9 - 1
Unique (20)
1 1 1 - - 1 - 12 1 - - - - - - 1 - 4
Total (62)
2 1 1 3 2 14 1 37 2 1 1 1 1 1 1 1 14 3 6
85
Table 4.3: Detection of Isoniazid Resistance mutations by katG and inhA gene sequencing of MDR-MTB strains
Genotype (N)
katG gene mutation
inhA gene mutation
Number of codon mutation Number of nucleotide mutation
315 agc acc
318 gag cag
352caa aaa
361gcc gtc
371cca tca
379gcc gtc
285ggc gtc
283ctg atg
274aag aac
251 acg aag
244gcg gag
242gcg
ggg
216 (C T)
CAS1 (30)
20 - - -
- - 1** 1 - 1 1 1 1
Beijing (12)
4* 1
1 1 1 - - - 2 - - - -
Unique (WHO)
13 - - -
- 1 - - - - - - -
Total (62) 37 1 1 1 1 1 1 1 2 1 1 1 1 *Denotes marginal significance of low occurrence of codon 315 mutation in Beijing strains than CAS1 and Unique (P= 0.052) ** Novel mutation
86
4.3: Results
4.3.1: Detection of rpoB gene mutations for Rifampicin resistance
Mutations in 495 bp region including 81bp hyper-variable region (RRDR)
of rpoB gene leading to rifampicin resistance were investigated. Using DNA
sequencing, mutations were detected in 56 (90%) of sixty two MDR strains
including 30 CAS1, 12 Beijing and 20 un-clustered spoligotyped strains.
Seventeen different combinations of mutations were identified in the rpoB gene of
MDR strains (Table 4.2). In addition mutations at codon 592 and 595 of rpoB
gene were noted. These however were seen in both resistant and susceptible
strains and therefore are not included in Table 4.2.
The mutations most commonly seen in MDR strains were in codons 531
(60%), 526 (23%) and 516 (5%) of rpoB gene. Most variability was seen in codon
526 with nine different combinations of mutations. A significantly higher
frequency of codon 526 mutation (93% vs. 7%, P= 0.008) was noted in CAS1 as
compared to Beijing and other un-clustered strains. Occurrence of more than one
mutation in rpoB gene was also significantly higher (40% vs. 12.5%, P= 0.014) in
CAS1 than Beijing and other un-clustered strains tested. Eighty-six percent of
total mutations were identified in RRDR region of the rpoB gene. 10% (n=6) of
the MDR strains tested did not show any mutations in the rpoB region.
87
4.3.2: Detection of mutations in katG and inhA genes for INH resistance
The presence of mutations in katG and inhA genes was explored further.
428 bp katG gene sequence covering codon 315 revealed twelve different sites of
mutations (Table 4.3). Sixty three percent (39/ 62) MDR isolates had a mutation
at codon 315. Our analysis (Table 4.3) revealed that CAS1 family strains
exhibited higher rate of mutation at codon 315 as compared with Beijing with
marginal significance (42% vs. 8%, P= 0.052). None of the mutations were
observed in the ten susceptible strains tested. Characterization of promoter region
of InhA gene revealed only one T-A mutation at nucleotide 216 of a CAS1 strain
as shown in Table 4.3.
88
4.5: Discussion
Our data reports commonly found mutations in the rpoB and katG genes of
predominant CAS1, Beijing and unique MDR strains. Overall the most affected codons
identified in rpoB gene were 531 (60%), 526 (23%) and 516 (5%). These findings are
comparable with previously reported data (Musser 1995). In agreement with previously
reported data from India (Siddiqi, Shamim et al. 2002) and China (Jiao, Mokrousov et al.
2007), all the mutations at codon 531 of rpoB gene were S531L. Occurrence of highest
frequency (67%) of mutation at codon 531 amongst the Beijing genogroups is also
comparable with data reported earlier (Banerjee, Dubnau et al. 1994; Sharma, Sethi et al.
2003).
Mutation at codon 526 of rpoB gene exclusively amongst the CAS1 strains
suggests an association between prevalence of this mutation and the CAS1 genogroup.
Codon 526 was furthermore found to be the most variable codon. Variability at this
codon has also been reported in a study from Russia (Afanas'ev, Ikryannikova et al.
2007). However our MTB isolates exhibited higher variablity at this codon as compared
to the data reported from other Asian countries (Hirano, Abe et al. 1999; Jiao, Mokrousov
et al. 2007). Our finding imply that 526 mutation results in no significant loss of fitness
and survival in CAS1 strains. Similar mutation and genogroup association was also
observed previously in a study where 75% of mutation at codon 516 of rpoB gene was
found amongst Latino-American and Mediterranean (LAM) genogroup (Lipin,
Stepanshina et al. 2007). Although studies from East Asian countries report 12%
89
mutation at codon 526 in Beijing family strains (Qian, Abe et al. 2002), we were unable
to confirm this finding.
In contrast, mutation at codon 516 was observed less frequently (5%) amongst our
isolates as compared to the data reported from other countries including those from Asia
(Hirano, Abe et al. 1999; Yuen, Leslie et al. 1999; Jiao, Mokrousov et al. 2007). Absence
of any mutation in the rpoB gene amongst ten percent of our MTB isolates is in
agreement with earlier reports (Telenti, Imboden et al. 1993; Hirano, Abe et al. 1999).
This finding may point towards presence of mutation in another region of rpoB or
presence of some other gene contributing towards rifampicin resistance.
Another important finding was the significant association of frequency of double
mutation (40%) with CAS1 genogroup. Although similar association has been reported
with Beijing genogroup strains from Latvia (Tracevska, Jansone et al. 2003), Beijing
strains in this study had not shown the same.
Overall, occurrence of mutation in codon 315 of katG gene correlates with the
range (60-90%) reported globally (Ramaswamy and Musser 1998). Although this,
frequency is not as high as reported from certain regions such as Russia (95%) and
Australia (91%) (Lavender, Globan et al. 2005; Lipin, Stepanshina et al. 2007). A
relatively high frequency of 315 mutation amongst our CAS1 isolates suggests that these
strains have an enhanced potential to retain peroxidase activity and decreased ability to
activate isoniazid by this mutation (Wengenack, Uhl et al. 1997). We further identified
one strain with single mutation at codon 285 (GGC-GTC) of katG that might be
responsible for isoniazid resistance. This mutation has not been described previously. In
contrast to katG gene, only one isolate showed mutation in the promoter region of inhA
90
gene. While most of the studies show around 15% of mutation in inhA gene (Baker,
Brown et al. 2005), relatively much lower frequency of this mutation amongst our
isolates may reflect local strain phenomena.
This study reports preliminary information regarding prevalent drug resistance
gene mutations amongst the MDR strains of predominant genogroups; CAS1 and Beijing,
from Pakistan. Data revealed that MDR CAS1 strains, which constitute half of the total
MDR strains in the country, were more prone to developing resistance against rifampicin
and isoniazid through mutations at codon 526 of rpoB gene and codon 315 of katG gene
respectively than Beijing and unique MDR strains. Within the MDR CAS1 strains 67%
of rifampicin and isoniazid resistance could be determined by detecting mutations at only
three codons 526 and 531 of rpoB gene and 315 of katG gene.
Knowledge of type and frequency of mutations amongst prevalent genotype
within defined geographical locations is essential for the development of appropriate
tools for early diagnosis and control of MDR-TB strains (Lipin 2007).A rapid diagnosis
of antituberculous drug resistance, especially RMP and INH, is particularly essential for
appropriate anti-tuberculous therapy and containment of the resistant strains
Therefore, using information of prevalent mutations for RMP and INH resistance
rapid molecular methods for MDR-TB detection amongst predominant genogroups;
CAS1 and Beijing were evaluated in the next part of this study.
91
Chapter Five
Molecular methods for rapid detection of Rifampicin and Isoniazid resistance amongst the MDR strains of predominant
genogroups of Mycobacterium tuberculosis 5.1: Background
It has been reported that the type and frequency of mutations varies for resistant
MTB strains within defined geographical locations (Lipin, Stepanshina et al. 2007).
Therefore distribution of rifampicin and isoniazid resistance mutations was determined in
the last part of this study using standard sequencing method (Chapter Four). Findings of
this study revealed rifampicin resistance mutation at codon 531 (60%), 526 (23%) and
Chapter preview Page #
5.1: Background 91 5.2: Objectives 94 5.3: Methods
5.3.1: Mycobacterial strains, Culture & DNA extraction 95 5.3.2: RMP and INH resistance detection using FRET 95 probe based Real Time PCR 5.3.3: RMP resistance detection using InnoLiPA 99 line probe assay 5.3.4: Statistical analysis 100
5.4: Results 5.4.1: Detection of mutation in rpoB gene for RMP 101
Resistance using FRET probes 5.4.2: Detection of mutation in katG & inhA genes 106 for INH resistance using FRET probes 5.4.3: Detection of rpoB RMP resistance by 106
INNO-LiPA assay 5.5: Discussions 109
92
516 (5%) of rpoB gene amongst the MDR-TB isolates. While for INH resistance, 63%
MDR isolates showed mutation at codon 315 of katG gene. These are important
preliminary findings for the development of rapid methods for detection of rifampicin
and isoniazid resistance in majority of MDR isolates from the country.
Different molecular assays have been proposed for the detection of mutations
associated with resistance to anti-TB drugs beside DNA sequencing which is considered
gold standard for molecular detection of drug resistance. These include real time PCR,
microarrays and kit based line probe assays (Torres 2002; Hillemann, Rusch-Gerdes et al.
2007). Use of rapid, simple and cost effective hybridization probe based real time PCR
method has been reported to detect between 60 -100 % mutations associated with
resistance to rifampicin and isoniazid (Torres 2002; Sajduda, Brzostek et al. 2004).
Limited data is available regarding use of this technique from within the Asian region.
Although a study based on isolates from India and Mexico reports detection rates of
100% (n=16) and 86% (n=64) respectively with the probe based method (Varma-Basil,
El-Hajj et al. 2004).
Commercially available kit based line probe assay (INNO-LiPA, Innogenetics
Zwijndrecht, Belgium) detects mutations in the rpoB gene for rifampicin resistance
(Rossau, Traore et al. 1997). The LiPA kit contains 10 oligonucleotide probes (one
specific for the M. tuberculosis complex, five overlapping wild-type S probes, and four R
probes for detecting specific mutations of resistant genotypes) immobilized on
nitrocellulose paper strips (Rossau, Traore et al. 1997). Since rifampicin resistance
93
usually develops in conjunction with isoniazid resistance reportedly in more than 90% of
rifampicin resistant isolates, this rapid and simple method could predict MDR (Traore,
Fissette et al. 2000). A number of studies have evaluated the diagnostic accuracy of LiPA
for the detection of rifampicin resistance in diverse geographic settings (Hirano, Abe et
al. 1999; Bartfai, Somoskovi et al. 2001; Johansen, Lundgren et al. 2003).
The aims of this study were to explore utility of two rapid molecular methods;
probe based real time PCR to identify common mutations present in rpoB, katG and inhA
genes and reverse hybridization Line probe assay (LiPA) to identify mutations in rpoB
gene amongst the prevalent genogroups; CAS1 and Beijing MDR-TB strains (identified
by sequencing, chapter four). Unique strains were also tested in order to compare the
results.
94
5.2: Objectives
The overall aim of this study was to evaluate a rapid test to detect rifampicin and
isoniazid resistance in prevalent MDR strains. Specific objectives were to
evaluate:
5.2.1: Real Time PCR method using Fluorescence resonance energy
transfer (FRET) probes for the detection of rifampicin and
isoniazid resistance in prevalent CAS1 and Beijing MDR-TB
strains from the country
5.2.2: InnoLiPA assay for the detection of rifampicin resistance in
MDR strains from prevalent genogroups
95
5.3: Methods 5.3.1: Mycobacterial strains, culture & DNA extraction
All 62 MDR-MTB and 10 susceptible strains which were used for
sequencing (Chapter Four) were selected for this study as well. These CAS1,
Beijing and un-clustered spoligotyped strains were cultured and DNA was
extracted by similar methods as described earlier in this study.
5.3.2: Rifampicin and Isoniazid resistance detection using FRET probe based Real Time PCR: (As described by Torres 2000 and Torres 2002)
Detection of mutation in rpoB katG and inhA genes was performed using
pairs of fluorescence resonance energy transfer (FRET) probes using Light Cycler
instrument (Torres, Criado et al. 2000; Torres, Criado et al. 2002). Briefly, FRET
probes consist of two probes anchor and sensor that are designed to hybridize
adjacent to each other on the complementary DNA sites. In this study 3’ end of
anchor probe was labeled with fluorescein, and the adjacent 5’end of the sensor
probe was labeled with Cal-635, another fluorescent dye (Figure 5.1). The Light
Cycler instrument activated fluorescein, which caused activation of the adjacent
dye, Cal-635. This resulted in emission of fluorescence at a different wavelength
(630-705). The detection of mutations within the DNA regions covered by the
FRET probes is based on the differential patterns of denaturation of the probes
which are bound either to homologous sequences or to sequences with a mutation.
The Tms in each of the cases will be different. Therefore, differences in the Tms for
96
the probes with respect to those obtained when assaying the probes with wt
sequences indicate the presence of a mutation in the DNA region covered by the
probes. Probes were labeled with fluorescein at the 3’ end and CAL-635 at the 5’
end. Probes and primers are listed in Table 5.1 for respective gene.
PCR reaction:
PCR reactants in a final volume of 20 μl included 2 μl of a commercial
ready-to-use reaction mix for PCR (Light-Cycler-DNA master hybridization
probes, Roche Diagnostics). MgCl2 was added to a final concentration of 4 mM.
The primers and probes were added to final concentrations of 1.4 and 0.3 μM,
respectively. 200ng of template DNA was used. The 20 μl (final volume) reaction
mix was placed in glass capillary cuvettes, which were filled by centrifugation in
a microcentrifuge.
PCR cycling condition:
Conditions for cycling were 95°C for 15 s, followed by 35 cycles of 94°C
for 1 s, 57°C for 45 s and 72°C for 30s. A melting program of 50 to 85°C at 0.1°
C/s with continuous monitoring of the fluorescence followed it. The Tm of each of
the FRET probes was calculated as the average value of the Tms obtained in at
least two independent experiments. The probe was homologous to the wild type
(wt) sequence of rpoB gene and led to different melting temperatures (Tms) when
hybridized with target DNA region with mutation. When deviations in Tm were
more than two standard deviations, a mutation was suspected.
97
Figure 5.1: Mechanism of action of FRET probes. A) Annealing step: the two FRET probes (anchor and sensor) bind to the template DNA. The head-to-tail positioning of the probes allows the two dyes to be close to each other. By a process of energy transfer, excitation of the anchor dye stimulates the sensor dye, which in turn emits fluorescence. B) The behavior of the sensor probe in the post-PCR melting step is shown for a wild-type and mutant sequence. The sensor probe is melted at lower temperature when a mutated sequence is found owing to a thermodynamically impaired binding of this probe. C) The presence of a mutation is detected by a deviation in the melting temperature of the probe. (Reference: Adapted from Garcia de Viedma 2003)
A
B
C
98
Table 5.1: Primers and FRET probes used for PCR amplification and detection of RMP and INH resistance in MTB strains
Target Gene
Primer or FRET Probe
Sequence Product size
RpoB rpoB F-primer TCGCCGCGATCAAGGAGT 158
rpoB R-primer GTGCACGTCGCGGACCTCCA rpoB probe sensor Cal635-ACCCACAAGCGCCGACTGCTGG-P rpoB probe anchor TTCATGGACCAGAACAACCCGCTGTCGGT-F
KatG katG F-primer GAAACAGCGGCGCTGATCGT 209
katG R-primer GTTGTCCCATTTCGTCGGGG katG probe sensor Cal635TCACCAGCGGCATCGAGGTCGT-P katG probe anchor CGTATGGCACCGGAACCGGTAAGGACGC-F
InhA inhA F-primer CCTCGCTGCCCAGAAAGGGA 248
inhA R-primer ATCCCCCGGTTTCCTCCGGT inhA probe sensor Cal635-CCCGACAACCTATCATCTCGCC-P inhA probe anchor CCCCTTCAGTGGCTGTGGCAGTC-F
Adapted from: (Torres, Criado et al. 2000; Sajduda, Brzostek et al. 2004)
99
5.3.3: Rifampicin resistance detection using InnoLiPA line probe assay
A subset of MDR CAS1 (n=26) and Beijing (n=7) were subjected to
INNO-LiPA analysis. Mycobacterium tuberculosis H37Rv strain was used as a
control. The hybridization assay was performed according to manufacturer’s
instructions. Brief account of the method is as follows:
PCR reaction:
The target DNA was amplified in a nested PCR using following
biotinylated primers and cycling conditions:
LIPAOP1 (outer primer), 59-GAGAATTCGGTCGGCGAGCTGATCC-39,
LIPA OP2 (outer primer), 59-CGAAGCTTGACCCGCGCGTACACC-39;
LIPA IP1 (inner primer), 59-GGTCGGCATGTCGCGGATGG-39; and
LIPA IP2 (inner primer), 59-GCACGTCGCGGACCTCCAGC-39.
The first-round PCR consisted of 30 cycles of 95°C for 60 s, 58°C for 30s, and
72°C for 90 s. The second-round PCR consisted of 30 cycles of 95°C for 20s,
65°C for 30 s, and 72°C for 30 s.
100
Probe hybridization:
Each PCR product was denatured and hybridized to membrane- bound
capture probes i.e. S-type probes (SI, S2, S3, S4 and S5) and four R- type probes,
which are specifically designed to hybridize to the sequences of the four most
frequently observed mutations; R2 (Asp-516-Val), R4a (His-526-Tyr), R4b (His-
526-Asp), and R5 (Ser-531-Leu).
Interpretation of the banding pattern:
The hybridization of the product of amplification with probes immobilized
on a nitrocellulose support was revealed by reaction of the biotine with the
streptavidine coupled with alkaline phosphate, which allowed detection of
respective mutation. (Rossau, Traore et al. 1997; Matsiota-Bernard, Vrioni et al.
1998; Watterson, Wilson et al. 1998; Hirano, Abe et al. 1999; Bartfai, Somoskovi
et al. 2001)
5.3.4: Statistical analysis
Association of a particular gene mutation with a genogroup was
determined by Fisher exact test using version 16 of SPSS (Special Program for
Social Sciences Software, USA). A p-value of < 0.05 was considered significant.
101
5.4: Results
5.4.1: Detection of mutations in rpoB gene for Rifampicin resistance using
FRET probes
Mutations in the rpoB, katG and inhA genes detected using FRET probes
are shown in Figure 5.2. The temperatures at which the probes melted (Tm) from
PCR products during the melting program are also shown. Tm was calculated
using the Light Cycler software. The average Tm for rpoB gene in ten susceptible
strains was 65.4 °C. Twenty seven (44%) MDR strains showed mutations in
codon 531 with an average >2 C increase while 6 (10%) strains showed
mutations in codon 526 with an average >2C decrease in the Tm of probe as
compared to the susceptible strains (Figure 5.1). Concurrent mutation of 531 and
526 in seven MDR strains resulted in 2 C increase in the Tm of melting probes.
The number of mutation detected in rpoB, gene of 62 MDR strains by probe
based real time PCR assay was 52. Sensitivity and Specificity of probe based
assay for detection of mutation in rpoB was 84% and 100% in comparison to
culture susceptibility testing and 93% and 100% respectively compared to
sequencing.
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Figure 5.2: Melting curves detecting rpoB mutation using FRET probes with three different types of samples. Rifampicin resistant MTB strains, with 531 codon mutation shows higher Tm while MTB strains with 526 codon mutation exhibit lower Tm compared to wild type MTB strain.
103
Table 5.2: Detection of prevalent Rifampicin and Isoniazid resistance by FRET probes using real time PCR Antimicrobial
agent Target gene
Mutation at codon(s)*/nucleotide
Number of strains
Mean Tm with standard deviation (°C )
Rifampicin rpoB Susceptible strains 10 65.4 0.29
531 27 68.81 1.21
531 + 526 07 67.46 0.64
526 06 63 1 516 03 65.82 0.79 531+522 02 68.9 0.14 513 01 67.2 531 + 527 01 67.2 526+ 511 01 62.2 515+511 01 65.1 No mutation 06 65.2 0.68
Isoniazid katG Susceptible strains 10 73.61 0.59 315 33 69.78 1.06 285 01 73.9 315 +242 01 74 315+379 01 69.18 315+352+274 01 70.7 315+383+351+244 01 69.4 318+361+371+274 01 71.3 No mutation 23 73.61 0.54 inhA Susceptible strains 10 64.76 0.74 216 nucleotide 01 59.7 No mutation 61 64.6 0.77
* Mutations arranged by decreasing occurrence
104
Figure 5.3: Melting curves detecting katG mutation using FRET probes with two different types of samples; wild type (WT) strains have perfect match to the sensor probe and a Tm of 73.6C; R type (INH resistant) MTB strains with the mutation at codon 315 have lower Tm 71.0C.
Figure 5.4: Melting curves detecting inhA mutation using FRET probes. Strains with wild type gene have a Tm of 64.6 C; similar to susceptible strains while only strain with a point mutation in the gene has almost 5 0C lower Tm.
105
Table 5.3: Summary of mutations identified by Sequencing and Real time PCR in Rifampicin and Isoniazid resistant MTB isolates as compared to phenotypic drug susceptibility testing (DST)
Geno-group
(N)
Rif resistance N (%)
INH resistance N (%)
Resistance
by DST rpoB gene mutation by
Resistance by DST
katG gene mutation
by
inhA gene mutation by
Sequenc-
ing
Real Time
Sequenc-
ing
Real Time
Sequenc-
ing
Real Time
CAS1(30)
30 29/30
(97)27/30 (87)
30
20/30 (66)
20/30
(66) 1/30 (3.3)
1/30 (3.3)
Beijing (12) 12 11/12
(92)9/12 (75)
12 5/12 (42)
5/12 (33) 0/12 0/12
Unique (20)
20 16/20 (85)
16/20 (80)
20 12/20 (60)
12/20
(55) 0/20 0/20 Concordan
ce With DST
56/62 (90)
52/62 (82)
37/62 (60)
37/62
(60) 1/62 (2)
1/62 (2)
106
Number of Strains
Mutation at rpoB codons
Figure 5.5: rpoB mutations identified by InnoLiPA assay
0
2
4
6
8
10
12
14
∆S1 ∆S2 ∆S1& S2
516 526 531 526&531
ND
CAS1
Beijing
107
Table 5.5: Comparison of results obtained by LiPA and DNA Sequencing of 33 MDR-TB strains
LiPA pattern
DNA sequencing result
# of isolates
CAS1 (n=26)
Beijing (n=7)
R5 ( Ser531Leu) TCGTTG ( Ser531Leu) 14 5 R2 ( Asp516Val) GACGTC (Asp516Val) 1 1 R4a ( His526Tyr) CACTCC ( His526 Ser) 3 0
R2 &R5 ( Asp516Val + Ser531Leu)
GACGTC (Asp516Val) TCGTTG ( Ser531Leu)
1 0
S1 CACCCC ( His526Pro) 1 0 S2 GACGTC (Asp516Val)
CACCCC ( His526Pro) 1 1
0 0
S1 & S2 CTGCCG (Leu511Pro) CACCCC ( His526Pro)
1 0
Wild type No mutation detected 3 1
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5.4.2: Detection of mutations in katG and inhA genes for isoniazid resistance using FRET probes
Average Tm for katG gene of ten susceptible strains was 73.6 °C. Thirty -
seven (60%) MDR strains showed mutations in codon 315 detected by a decrease
of > 3C of temperature in probe’s Tm as compared to the susceptible strains
(Figure 5.2). One mutation in the promoter region of inhA gene was also detected
using inhA hybridization probes, with decrease of 5 C in the Tm as compared to
the 64.7 C Tm of susceptible strains (Figure 5.3). The number of mutations
detected in katG and inhA genes of 62 MDR strains by probe based real time PCR
assay was 37 and 1 respectively. Sensitivity and Specificity for detection of
mutation in katG was 60% and 100% in comparison to culture susceptibility
testing and 95% and 100% respectively compared with sequencing.
5.4.3: Detection of Rifampicin resistance by INNO-LiPA assay
Rifampicin resistance mutations were detected by LiPA in 29/33
rifampicin-resistant MTB strains. Nineteen MTB isolates showed S531L, three
isolate had H526Y, and two isolated showed D516V mutation while five strains
showed mutations other than these codons (Figure 5.4).
LiPA results were also compared to culture susceptibility and sequencing
as well. The sensitivity and specificity of the LiPA assay for detection of rpoB
mutation in comparison with culture susceptibility testing and sequencing was
88% and 100% respectively. Overall 29/33 resistant MTB strains were found
resistant by LiPA assay. The resistant strains, which were not detected by LiPA
were also found to be wild type by sequencing (Table 5.3).
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3.6: Discussion
Using the information of common mutations for rifampicin and isoniazid
resistance (discussed in Chapter Four) previously described FRET probe based real time
PCR method was evaluated for the detection of mutations in rpoB, katG and inhA genes
(Torres, Criado et al. 2000; Torres 2002).
Using rpoB probes, mutations were detected in 84% of the MDR isolates. Overall
the frequency of mutations identified were in agreement with data reported earlier (Torres,
Criado et al. 2000; Garcia de Viedma 2003; Sajduda, Brzostek et al. 2004). These
findings supports that probe based assay may be useful as a rapid diagnostic tool for
detection of rifampicin resistance.
While using katG probe, mutation in 60% of the isoniazid resistant isolates was
detected at codon 315 similar to that of sequencing. Detection of mutation at codon 315 is
clinically important, as it has been linked with high level of isoniazid resistance (van
Soolingen, de Haas et al. 2000; Marin, Garcia de Viedma et al. 2004). Additionally katG
315 mutations have also been found to be associated with successful transmission of
MDR-TB within the population (van Soolingen, de Haas et al. 2000). Thus, the utility of
katG probes for detection of this mutation in this geographical location may be vuseful
for rapid detection of MDR-TB strains.
110
With inhA probes only one mutation was detected which was in accordance with
sequencing results. This finding is in contrast to the study from Equatorial Guinea in
which none of the mutations were observed in katG gene while 80.5% mutations were
detected in inhA gene (Tudo, Gonzalez et al. 2004). Thus, low prevalence of mutation in
inhA gene of MDR strains from this country might be a regional phenomenon, which
needs further investigation.
LiPA assay has been used in several studies for the rapid detection of resistance to
rifampicin in MTB isolates. These studies have shown greater than 95% of sensitivity and
specificity (Morgan, Kalantri et al. 2005) and allowed prediction of MDR in more than
95% of the cases (Traore, Fissette et al. 2000). In this study LiPA assay showed 88% and
100% of sensitivity and specificity respectively. High (100%) specificity and rather lower
sensitivity has also been reported in other studies (Morgan, Kalantri et al. 2005; Traore,
van Deun et al. 2006). The high specificity suggests that LiPA test is a good predictor for
MDR and may be used in this setting for detection of rifampicin resistance from majority
of resistant isolates. The cost of the test on the hand is the major limitation for the
application of this test at large (Piersimoni and Scarparo 2003).
In conclusion, FRET probe based methods are less expensive and rapid, and have
the potential of detecting wide variety of resistance mutations in majority of clinical
MTB isolates in our geographical area. Use of this method in conjunction of routine
susceptibility testing for MDR-TB detection might have major impact on the
management of multidrug-resistant tuberculosis.
111
Chapter six
General discussion and Conclusions
World Health Organization declared tuberculosis as a global emergency in
1993. Increase in population, migration and high incidence of immuno deficiency
virus (HIV) infection are major contributory factors for the resurgence and spread of
tuberculosis in the late 20th and early 21st century. The understanding of tuberculosis
transmission dynamics has been greatly enhanced by the availability of genotyping
tools. Using appropriate genotyping methods molecular epidemiological studies of
Mycobacterium tuberculosis have provided novel insights into the biogeography of
tuberculosis, which has been shown to play a significant role in proposing new and
innovative strategies for control of tuberculosis.
Pakistan accounts for approximately half of the total tuberculosis cases in the
Eastern Mediterranean region with estimated number of 423000 cases. This figure
implies that a large number of people in the country are serving as disease reservoir.
Additionally, approximately 75% of the infected cases are in the productive years
(15-59) of their life, the illness exerting an additional economical burden on their
families and on the country at large (WHO 2008). Moreover, non compliance and
premature cessation of treatment by one out of every four-five tuberculosis patients
results in development of drug resistant tuberculosis (Liefooghe, Michiels et al. 1995).
According to recent estimate prevalence of MDR-TB amongst untreated cases in
Pakistan is 1.8% (Javaid, Ghafoor et al. 2008).
112
This study provides first insight into the genetic differences of
Mycobacterium tuberculosis, including Central Asian Strain1 (CAS1), identified as a
predominant genogroup from Pakistan (Hasan, Tanveer et al. 2006). CAS1 family
strains investigated in this study are a closely related strain type of Mycobacterium
tuberculosis and are wide spread in South Asian countries including India and
Bangladesh (Banu, Gordon et al. 2004; Bhanu, Banavalikar et al. 2004). In addition
these strains have also been reported from Sudan and were also found amongst South
Asian immigrants in England (Gascoyne-Binzi, Barlow et al. 2002; Brudey, Driscoll
et al. 2006). Thus it is important to carry out in depth molecular studies using more
discriminatory methods than spoligotyping to study these strains further.
This study used MIRU-VNTR and IS6110-RFLP molecular typing methods
to unravel intra-strain differences amongst CAS1 strains from the country. IS6110-
RFLP and MIRU-VNTR typing revealed highly discriminative profiles with
relatively lower clustering of MTB strains from this geographical setting. This
relatively high genetic diversity in MTB population including CAS1 strains was
unexpected, since studies have demonstrated lower levels of genetic diversity in high-
incidence communities (Pineda-Garcia, Ferrera et al. 1997; Easterbrook, Gibson et al.
2004; Verver, Warren et al. 2004; Nikolayevskyy, Gopaul et al. 2006). The diverse
genotypic profiles might support the hypothesis that majority of cases in the country
arise from endogenous reactivation of diverse latent tuberculosis infection as opposed
to cross transmission. Such high genetic diversity has also been reported from other
113
countries including India and Bangladesh and some African regions (Tazi, Reintjes et
al. 2007; Hanekom, van der Spuy et al. 2008; Sharma, Kalyani et al. 2008).
There may be three major reasons for the high genetic polymorphisms in the
MTB strains from the country; diverse host population with different ethnicity,
ancient TB endemicity and genetic variability. Studies have suggested co-evolution of
the MTB strains with diverse population over a long period of time in any
geographical region (Brosch, Gordon et al. 2002; Gagneux, DeRiemer et al. 2006).
Pakistan; part of Indian Subcontinent, is an old endemic TB region. Millions of
people have migrated to Pakistan from India after the partition of Subcontinent in
1947. Many of them must have brought strains from different parts of the
subcontinent. Besides, Pakistan also faced large number of influxes of immigrants
after the liberation of Bangladesh in 1971 and after Soviet Union invasion of
Afghanistan in 1979 (Meulemans 2000). It has been proposed that TB endemicity
and host-pathogen coexistence in a population requires a social network of
approximately 200-400 persons (McGrath 1988). In our geographical location many
communities may have experienced the exposure of fundamental genotypes of
Mycobacterium tuberculosis in past. Thus these fundamental MTB genotypes may
have had ample time to create a large number of population adapted genetic variants
(Sola, Filliol et al. 2001; Gagneux, DeRiemer et al. 2006). High genetic diversity may
also be due to genetic variation resulting from insertion/deletion events (Brosch,
Gordon et al. 2002).
114
Studies have grouped CAS1 strains under ‘modern’ MTB lineage due to absence
of Mycobacterium tuberculosis specific ‘TbD1 region’ (Sreevatsan, Pan et al. 1997;
Brosch, Gordon et al. 2002; Gutierrez, Ahmed et al. 2006). It has also been suggested
that the clones which take longer to evolve result in more extensive genetic diversity
(Sola, Filliol et al. 2001). In addition, Region of difference (RD) 750 has also been
reported deleted from CAS family strains. This deletion has been suggested to be
involved in the lower production of protective cytokines (Gagneux, DeRiemer et al.
2006; Newton, Smith et al. 2006; Gagneux and Small 2007), which might had a role
in persistence and spread of CAS1 strains in the region.
Beside genetic diversity MIRU-VNTR typing also revealed seven most
discriminatory MIRU loci i.e. 26, 31, 16, 10, 27, 39 and 40 for the molecular
epidemiological studies in the geographical area where CAS1 strains are common.
Five of these seven loci identifies (26, 31, 10, 39 and 40) were also the most
discriminatory in other studies (Mazars, Lesjean et al. 2001; Supply, Warren et al.
2003), suggesting that the relative degree of information carried by the different loci
are globally conserved in MTB population. These limited numbers of polymorphic
loci demonstrate high discriminatory value for the prevalent MTB population from
this region and thus could be used as a cost effective molecular typing method.
Application of limited number of loci may be a useful and rapid genotyping tool for
studying transmission dynamics in the population where CAS1 strains are prevalent.
Study from our lab also revealed that almost half of the predominant CAS1 strains
comprise of MDR (Hasan, Tanveer et al. 2006). In addition, drug resistance in MTB
115
has been shown to result from acquisition of resistance mutations within the
chromosomes (Sreevatsan, Pan et al. 1997). Therefore mutations in the drug
resistance genes for MDR amongst the predominant genogroups were also
investigated. Results from this study suggest that more than 60% of rifampicin and
isoniazid resistance within MDR CAS1 strains could be determined by detecting
mutations at only three codons 526 and 531 of rpoB and 315 of katG gene. Study
further revealed that MDR CAS1 strains were more prone to develop resistance
against rifampicin and isoniazid through mutations at codon 526 of rpoB gene and
codon 315 of katG gene respectively as compared to non-CAS1 MDR strains. This
data is in contrast to the study suggesting an association between codon 526 mutation
and a decrease in fitness (Gagneux, Long et al. 2006). Predominance of CAS1 MDR
strains in our population on the other hand suggests that 526 mutation does not result
in any significant loss of fitness and survival in CAS1 strains. This implication is also
supported by an earlier study where six out of seven CAS1 MDR strains had
mutations at codon 526 of rpoB gene (Githui, Jordaan et al. 2004).
For isoniazid resistance CAS1 isolates showed relatively higher frequency of
codon 315 katG gene mutation compared to Beijing strains. Similar findings have
also been reported previously in a study, which noted codon 315 mutation amongst
six out of seven CAS1 MDR strains (Githui, Jordaan et al. 2004). There is a
possibility that with codon 315 katG mutation CAS1 strains have an enhanced
potential to retain peroxidase activity and decreased ability to activate INH by this
mutation as suggested by earlier studies (Wengenack, Uhl et al. 1997; Baker, Brown
116
et al. 2005). On the other hand, studies have suggested an inverse relationship
between propagation of resistant MTB strains and other than codon 315 katG
mutations in a defined geographical location (Pym, Saint-Joanis et al. 2002; Gagneux,
Burgos et al. 2006). Thus, significantly higher katG mutations in codons other than
315 in Beijing strains might explain comparatively lower prevalence of Beijing
strains than CAS1 in this geographical setting. However, further investigation is
required to validate this implication.
In addition, for the development and implementation of rapid MDR detection
amongst MTB stains circulating within Pakistan probe based real time PCR methods
have shown promising results. Using rpoB, katG and inhA Florescence Resonance
Energy Transfer probes (FRET), common mutations in 84% and 60% of the
rifampicin and isoniazid resistant respectively isolates were detected. These data
suggest that probe based assay may be useful as a rapid diagnostic tool. However
findings from this study further show that reliance on probe based assay alone will be
unable to detect all the resistance cases and that such analysis will require to be
complemented with routine susceptibility testing for MDR detection. In future probe
based real time PCR method could be tested on larger population of MTB strains.
Additionally, method needs to be compared with recently introduced line probe
assays for the detection of resistance in MDR-TB strains in terms of cost and time in
order to make recommendations for its application in detection of rifampicin and
isoniazid resistance (Hillemann, Rusch-Gerdes et al. 2007).
117
In conclusion, molecular typing techniques established in this study, could enhance
understanding of transmission dynamics of disease and could be used to improve
current control programs for tuberculosis in the country. Community based studies
using these established techniques may shed more light on the transmission of
susceptible and drug resistant MTB strains by tracking these strains in the community.
Genotyping information of MTB strains together with epidemiologic investigations
may provide important information about the spread of MTB strains by identifying
factors related to transmission and progression to tuberculosis disease. This in turn
could greatly assist in formulating strategies for control of tuberculosis. The data
obtained in this study further demonstrates that use of rapid drug resistance detection
methods in conjunction of routine susceptibility testing, such as FRET probe based
method used in this study for MDR-TB detection, could further assist in the
management of multidrug-resistant tuberculosis in the country by timely and adequate
use of anti-tuberculosis therapy. As Ian Sutherland noted: “one man’s cure is many
men’s prevention” (Meulemans 2000).
118
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Appendices