multi-drug resistant Tuberculosis mg milligram. MgCl2. Magnesium chloride. MMR ... gram kbp kilo base pare min minute ml millilitre. mM millimolar sec seconds μg ..... products either activate the drug or in turn are the drug target (145). ...... 0.7824. 160. 160. 2.7550. Taq 590. GG. GA. AA. 59. 30. 3. 60. 29. 3. 89. 54. 17. 84.
RISK FACTORS ASSOCIATED WITH ISONIAZID RESISTANCE IN TUBERCULOSIS
MARINUS BARNARD
Thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Medical Sciences (Medical Biochemistry) at the University of Stellenbosch
Promoter: Prof Thomas C. Victor Co-promoter: Prof Rob M. Warren December 2005
You know, Tolstoy, like myself, wasn’t taken in by superstitions – like science and medicine. - George Bernard Shaw
i
DECLARATION
I, the undersigned, hereby declare that the work contained in this thesis is my own original work and has not previously, in its entirety or in part, been submitted at any university for a degree.
Signature
Date
ii
SUMMARY
Tuberculosis (TB) is one of the most serious infectious diseases known to mankind, with devastating outcomes in the poorest countries in the world. Isoniazid is the cornerstone of all first-line anti-TB regimens. Forty-eight percent of all drug resistant TB isolates in the Western Cape are Isoniazid mono-resistant, and the majority of these isolates belong to the Beijing/W strain family. Currently, the known molecular mechanisms which confer Isoniazid resistance in these isolates are attributed to mutations within the katG gene and account for up to 70% of all drug resistant TB isolates. Risk factors for the development of Isoniazid resistance can be attributed to either pathogen or host related factors and may partially account for the other 30% of Isoniazid resistant isolates.
In this study, three aspects which may
contribute to Isoniazid resistance were
investigated: DNA repair in the bacterium, host response to anti-TB treatment and socioeconomic factors.
A PCR based dot-blot strategy was used to screen for previously reported missense mutations in the mutT2, Rv3908 and ogt DNA repair genes of different strains of M. tuberculosis. All the Beijing isolates (drug resistant and susceptible), in contrast to the Atypical Beijing strains and other dominant strain families, exhibited missense mutations in all three base excision repair genes. It is therefore speculated that defects in the DNA repair genes (mutator phenotypes) of the Beijing isolates may contribute to the
iii development of drug resistance and hence, may account for the large proportion of isolates that are Isoniazid mono-resistant.
A novel method, based on primer extension, was initially developed to screen the NAT2 gene and then used to type individuals into fast, intermediate and slow acetylators of Isoniazid. The newly develop method, which is sensitive and accurate, improves the detection of Single Nucleotide Polymorphisms within the NAT2 gene, in contrast to the traditionally used methods. Utilising this method, it was found that the combination of fast and intermediate acetylators was significantly associated with Isoniazid resistance in the study community.
This finding may have an important impact on TB control
programmes, since it may allow for the administration of higher dosages of Isoniazid to fast/intermediate acetylators and a lower dose for slow acetylators.
Clinical factors (compliance and retreatment after cure) and socio-economic factors (education, employment and income) were found to be significantly associated with the development of INH resistance. Diagnostic delay was also found to be a risk factor, since it may allow for transmission of TB during this period. The HIV prevalence in the study population is low and subsequently HIV status was not associated with the development of INH resistance.
This study indicates that a combination of risk factors, both pathogen and host related, are involved in the development of Isoniazid resistance.
iv
OPSOMMING
Tuberkulose (TB) is een van die ernstigste infektiewe siektes bekend aan die mensdom en die uitkoms is veral sleg in die armste lande. Die basis van eerste-linie anti-TB behandeling is Isoniasied. In die Wes-Kaap is 48% van alle middel-weerstandige TB isolate Isoniasied mono-weerstandig en die meerderheid van hierdie isolate behoort aan die Beijing/W familie. Op die huidige oomblik kan die molekulêre meganismes wat Isoniasied weerstandigheid bemiddel, toegeskryf word aan mutasies in die katG geen en hierdie meganisme is verantwoordelik vir amper 70% van alle middel-weerstandige TB isolate. Risiko faktore vir die ontwikkeling van Isoniasied weerstandigheid kan toegeskryf word aan of patogeen- of gasheer-verwante faktore en mag gedeeltelik vir sowat 30% van middel weerstandige isolate verantwoordelik wees.
In hierdie studie is drie faktore wat kan bydra tot Isoniasied weerstandigheid ondersoek: DNS herstel in die bakterium, die gasheer se reaksie op anti-TB behandeling en sosioekonomiese faktore.
‘n PKR gebasseerde “dot-blot” benadering was gebruik om te sif vir bekende mutasies in die mutT2, Rv3908 en ogt DNA herstel gene van verskillende stamme van M. tuberculosis. Alle Beijing isolate (middel-weerstandig sowel as -vatbaar) het wel hierdie mutasies in al drie basis-eksisie herstel gene getoon in teenstelling met die Atipiese Beijing stam en ander dominante stam families.
Daar word dus gespekuleer dat
defektiewe DNS herstel gene (mutator fenotipes) van die Beijing isolate kan bydra tot die
v ontwikkeling van weerstandigheid en om die rede, mag dit verantwoordelik wees vir die hoë proporsie van isolate wat Isoniasied mono-weerstandig is.
‘n Nuwe metode, wat gebruik maak van volgorde ekstensie, was inisieël ontwikkel om die NAT2 geen te sif en toe gebruik om persone diensooreenkomstig te tipeer as vinnige, intermediêre en stadige asetileerders van Isoniasied. Die nuut ontwikkelde metode, wat sensitief en akkuraat is, bevorder die sifting van Enkel Basispaar Polimorfismes in die NAT2 geen in teenstelling met die tradisioneel gebruikte metodes. Met hierdie metode is gevind dat in die studie gemeenskap, die gekombineerde groep van alle vinnige en intermediêre asetileerders betekenisvol geassosieerd was met weerstandigheid teen Isoniasied. Hierdie bevinding kan dalk belangrike gevolge inhou vir huidige TB beheer programme, deurdat hoër dossise van Isoniasied aan pasiënte wat vinnige/intermediêre asetileerders is voorgeskryf kan word en ‘n laer dosis vir stadige asetileerders.
Dit is bevind dat kliniese faktore (getroue middelgebruik en herbehandeling na genesing) sowel as sosio-ekonomiese faktore (opvoeding, inkomste en nering) betekenisvol geassosieerd was met die ontwikkeling van Isoniasied weerstandigheid. ‘n Vertraging in diagnose is ook ‘n risiko faktor, synde dat daar TB oordrag kan plaasvind gedurende hierdie periode. Die populasie groep het ‘n lae MIV insidensie gehad en dus was MIV status nie met die ontwikkeling van Isoniasied weerstandigheid geassosieer nie.
Hierdie studie toon dat ‘n kombinasie van risiko faktore, patogeen- en gasheerverwant, betrokke is in die ontwikkeling van Isoniasied weerstandigheid.
vi
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to the following people who have assisted me during the writing of this thesis:
To Prof. Tommie Victor, thank you for trusting me with this project and for allowing me to work in the laboratories at Medical Biochemistry. Many thanks also for the financial support in all aspects of this study. To Prof. Rob Warren, my deepest appreciation for all your time, effort, availability and enthusiasm during all aspects of this study.
I would also like to thank the following people, in no specific order: •
Ms. Annemie Jordaan, for levity and support.
•
The people in Lab 453.
•
Sr Susan Maass, for help with recruiting patients.
•
Mrs. Marianne de Kock for enormous help in experimental procedures.
•
Mr. Cedric Werely for informative discussions.
•
Prof. Megan Murray for constructive input.
•
Dr. Lize van der Merwe for invaluable help with statistical analysis.
•
Lovie, my mother earth.
•
Chakotay, Scampy and Bok.
vii
ABBREVIATIONS A
adenosine
Ac-CoA
acetyl Coenzyme A
Ac.INH
acetyl isoniazid
ACP
acyl carrier protein
A.D.
anno domini
AIDS
acquired immune deficiency syndrome
Ala
alanine (A)
Arg
arginine (R)
AS-PCR
allele specific PCR
BER
base excision repair
BLAST
Basic Local Alignment Search Tool
BOKS
Boland/Overberg – Southern Cape/Karoo
C
cytosine
CDC 1551
Mycobacterium tuberculosis clinical strain
CI
Confidence interval
CS
Cycloserine
Cys
cysteine (C)
DDR
direct damage reversal
DNA
deoxyribonucleic acid
dNTP
deoxynucleoside triphosphate
ddNTP
dideoxynucleoside triphosphate
DOTS
Directly Observed Treatment Short-course
DR
direct repeat
DRF 150
drug resistant family 150
DSB
double stranded breaks
DST
drug susceptibility testing
E. coli
Escherichia coli
EDTA
ethylene diamine tetra-acetic acid
ELISA
enzyme-linked immunosorbent assay
EMB
Ethambuthol
EtBr
ethidium bromide
viii F11
Family 11
F28
Family 28
F29
Family 29
G
Guanine
Gln
glutamine (Q)
Glu
glutamate (E)
Gly
glycine (G)
H
hydrogen
HCl
hydrochloric acid
HIV
Human Immunodeficiency Virus
H. pylori
Helicobacter pylori
HWE
Hardy-Weinberg equilibrium
H2O
Water
H37Rv
Mycobacterium tuberculosis laboratory strain
Ile
isoleucine (I)
INH
Isoniazid
IS
insertion sequence
IUATLD
International Union Against Tuberculosis and Lung Disease
L
litre
LCC
Low copy number clade
Leu
leucine (L)
LJ
Lowenstein-Jensen
Lys
lysine (K)
M
molar
M. avium
Mycobacterium avium
MDR
multi-drug resistant
MDR-TB
multi-drug resistant Tuberculosis
mg
milligram
MgCl2
Magnesium chloride
MMR
mismatch repair
mRNA
messenger RNA
M. smegmatis
Mycobacterium smegmatis
M. tuberculosis
Mycobacterium tuberculosis
ix N
nitrogen
NaCl
sodium chloride
NADH
reduced nicotinamide adenine dinucleotide
NaOH
sodium hydroxide
NAT
N-acetyltransferase
NCBI
National Centre for Biotechnology Information
NER
nucleotide excision repair
ng
nanograms
NHLS
National Health Laboratory Services
nm
nanometers
nt
nucleotide
O
oxygen
OD
Optical density
OR
Odds ratio
ORF
open reading frames
PBS
phosphate-buffered saline
PCR
polymerase chain reaction
PE
primer extension
PGG
principal genetic group
pmol
picomolar
PZA
Pyrazinamide
RER
recombinational repair
RFLP
Restriction Fragment Length Polymorphism
RIF
Rifampicin
RNA
ribonucleic acid
RNI
reactive nitrogen intermediates
ROI
reactive oxygen intermediates
rpm
revolutions per minute
RR
resistance ratio
SA
South Africa
SAP
Shrimp Alkaline Phosphatase
SDS
sodium dodecyl sulfate
Ser
serine (S)
SNP
Single Nucleotide Polymorphism
x STR
Streptomycin
T
Thymine
TB
Tuberculosis
TBE
tris-boric acid-ethylene diamine tetra-acetic acid buffer
Th2
T helper type-2
Thr
threonine (T)
TIGR
The Institute for Genomic Research
Tm
melting temperature
Tris
Tris(hydroxymethyl)aminomethane
U
units
USD
united states dollars
UV
ultra violet
Val
valine (V)
V
volts
WHO
World Health Organisation
w/v
weight per volume
ZAR
South African rands
ZN
Ziehl-Neelsen
bp
base pair
cm
centimeter
g
gram
kbp
kilo base pare
min
minute
ml
millilitre
mM
millimolar
sec
seconds
μg
microgram
μl
microlitre
μm
micrometer
μM
micro molar
°C
degrees Celsius
χ2
Chi square
xi /
per
5’
5 prime end
3’
3 prime end
%
percent
~
approximately
more than
γ-32P
gamma Phosphorus 32
σ
sigma
P
xii
LIST OF FIGURES PAGE Chapter 1 Figure 1:
Estimated Global TB Incidence Rates in 2002
4
Figure 2:
Metabolic Pathway of NAT2
37
Chapter 2 Figure 1:
Dendogram of IS6110 RFLP of Representative Isolates from Different M. tuberculosis Strain Families
76
Figure 2:
Conformation of PCR Amplification
77
Figure 3:
Dot-Blot Mutational Analysis
78
Figure 4:
Schematic Diagram Illustrating a possible Evolutionary Scenario based on the Acquisition of Missense Mutations in the DNA Repair Genes in the Beijing Strain Family of M. tuberculosis
79
Chapter 3 Figure 1:
Schematic Illustration of the most Frequently Observed NAT2 Allelic Variants.
Figure 2:
93
Illustration of Electropherograms of Individual 8 for Multiplex 1 (Figure 2A) and multiplex 2 (Figure 2B)
94/95
Figure 1:
Schematic Illustration of the NAT2 Allelic Variants
114
Figure 2:
Conformation of PCR Amplification of the NAT2 Gene
115
Age Distribution of the three Study Groups
143
Figure 1:
Illustration of the Three Putative DNA Repair Genes
159
Figure 2:
The SNaPshot Primer Extension Genotyping Method
167
Chapter 4
Chapter 5 Figure 1:
Chapter 6
xiii
LIST OF TABLES PAGE Chapter 1 Table 1:
Critical Drug Concentrations for Routine Susceptibility Testing
Table 2:
Cellular Targets for Anti-mycobacterial Drugs and Gene Mutations responsible for Resistance in M. tuberculosis
Table 3:
19
Homologues of E. coli Genes found within M. tuberculosis, involved in DNA repair
Table 4:
10
29
Most Common Nucleotide and Amino Acid Substitutions found in the NAT2 Allele Clusters
40
Table 1:
Representative Clusters of Isolates per Strain Family Selection
73
Table 2:
Primer Sequences used in the Amplification of the Three Putative
Chapter 2
Mutator Genes in M. tuberculosis
74
Table 3:
PCR Conditions for the Three Mutator Genes in M. tuberculosis
74
Table 4:
Probes for Dot-Blot Hybridization
75
Table 5:
Genome Comparison of the Reference Strain H37Rv versus the Clinical Strain CDC 1551 and the Beijing Strain M. tuberculosis 210
75
Chapter 3 Table 1:
Primers for initial Amplification of the NAT2 Gene and Primer Extension Multiplex Primers
91
Primer Extension versus combined RFLP and AS-PCR analysis
92
Table 1:
Hardy-Weinberg Equilibrium Calculations for each of the 7 SNPs
116
Table 2:
Distribution of the Acetylator Status in the Study Groups
117
Table 3:
Post hoc 2 x 2 Contingency Table applied to the Combined Acetylator
Table 2:
Chapter 4
Table 4:
Status between the Study Groups
117
Outcome of Compliant Patients Infected with the Beijing Strain
117
xiv Table 5:
Distribution of Acetylator Status between Patients Infected with Unique Strains and Controls
118
Table 1:
Time for MDR-TB Drug Resistance Diagnosis
143
Table 2:
Clinical Characteristics of Patients Infected with the
Chapter 5
Beijing Susceptible Strains and Beijing Resistant Strains according to Resistance Phenotype Table 3:
Table 4:
144
Clinical Characteristics of Patients Infected with the most Prominent Clusters within the Beijing Strain
145
Socio-Demographic Characteristics of Cases and Controls
146
Chapter 6 Table 1:
Primers used for PCR and Sequencing of the Three Putative DNA Repair Genes
Table 2:
158
Wild-type and Mutant Specific Probes used for Dot-Blot Hybridisation
159
Table 3:
Primers used for initial PCR Amplification of the NAT2 Gene
160
Table 4:
Primers used for the SNaPshot Primer Extension Method
161
Table 5:
PCR Conditions
162
xv
LIST OF SUPPLIERS Absolute Ethanol
Merck
Agarose (Seakem)
Sterilab
Boric Acid
Merck
Denhardt’s D9905
Sigma
Developer
Sigma
DNA marker (PhiX174)
Promega
dNTPs (GATC)
Promega
EDTA
Sigma
Eppendorf tubes
Merck
Ethidium Bromide
Sigma
Fixer
Sigma
HotStarTaq
Qiagen
Nylon Transfer Membrane Hybond N+
Amersham
p32 ATP
Amersham
Primers and Probes
Whitehead
Phenol
Merck
SDS
Sigma
Sodium Chloride
Merck
Sodium hydrogen phosphate
Merck
Sodium Hydroxide
Merck
T4-kinase
Amersham
Tris
ICN Biochemicals
Whatman 3m Paper
Merck
X-ray Film x-Omat
Sigma
Loading Buffer
Sigma
Contrad Radio active decontaminant
Merck
Dark room / Hyperprocessor RPN 1700
Amersham
Dot-Blot Apparatus
Biorad
Electrophoresis Documentation System 120Kodak Digital Science
Life Technologies
xvi Gel Electrophoresis System horizontal
Sigma
Heating Block Digi-block
Sigma
Hybridization Oven
Stuart Scientific
Laminar Flow Class II
Sigma
Orbital Shaker
Sigma
PCR Machine Mastercycler ep-gradient S
Eppendorf
pH-meter
Sigma
Pipettes Gilson
Sigma
Power Pack 300
Biorad
Radioactive Monitor
Amersham
Radio-active Waste disposal container
Sigma
Radio-active Workshield
Sigma
Vacuum Pump
Biometra
Vortex Mixers
Sigma
Heat-Sealer for plastic during hybridization
Sigma
x-Ray Cassettes
Sigma
Nucleon 3 DNA Extraction Kit
Amersham
SNaPshot Primer Extension Kit
Applied Biosystems
INDEX PAGE Declaration
i
Summary
ii
Opsomming
iv
Acknowledgements
vi
Abbreviations
vii
List of Figures
xii
List of Tables
xiii
List of Suppliers
xv
CHAPTER 1: LITERATURE REVIEW
1
INTRODUCTION HISTORY OF TUBERCULOSIS
2
IMPACT OF TUBERCULSOSIS
3
TB Control
5
Drug Resistance
6
Drug Susceptibility Testing
9
MOLECULAR EPIDEMIOLOGY
11
Definition of the Beijing/W Strain of M. tuberculosis
13
MOLECULAR MECHANISM OF ISONIAZID RESISTANCE
15
Permeability of the Cell Wall and Isoniazid Resistance
15
Drug Resistance Conferring Mutations
17
The Catalase-Peroxidase Enzyme and Isoniazid Resistance
20
Mutations within the KatG Gene
20
Increased Expression of Target Proteins The Enoyl Reductase Enzyme and Isoniazid resistance
22 23
The Alkylhydro-Peroxidase Enzyme and Isoniazid Resistance
23
The B-Ketoacyl Syntahse Enzyme and Isoniazid Resistance
24
The NADH Dehydrogenase Enzyme and Isoniazid Resistance
24
The OxyR Enzyme and Isoniazid Resistance
24
POSSIBLE ROLE OF DNA REPAIR IN THE DEVELOPMENT OF DRUG RESISTANCE
25
DNA Repair in M. tuberculosis
26
Base Excision repair
30
SOS Repair and Mutagenesis
30
Adaptive Mutagenesis
31
Hypermutators
32
Selection for Mutator Phenotypes
34
POSSIBLE ROLE OF THE HUMAN N-ACETYLTRANSFERASE 2 GENE IN THE DEVELOPMENT OF ISONIAZID RESISTANCE
35
Molecular Mechanism of the human N-Acetyltransferase Enzyme
36
Activation of Isoniazid by N-Acetyltransferase
38
N-acetyltransferase Genes
38
Human Acetylation Polymorphisms
39
Variation in Slow Acetylation Allele Frequencies
42
Mycobacterial N-Acetyltransferase and Isoniazid Resistance
44
SOCIO-ECONOMIC FACTORS WHICH MAY PLAY A ROLE IN ISONIAZID RESISTANCE
44
HYPOTHESIS
49
Overall Aim
49
CHAPTER 2: MYCOBACTERIAL DNA REPAIR AND ISONIAZID RESISTANCE
51
Introduction
54
Materials and Methods
59
Results
66
Discussion
69
CHAPTER 3: PRIMER EXTENSION IMPROVES THE DETECTION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE HUMAN N-ACETYLTRANSFERASE 2 GENE
84
CHAPTER 4: POSSIBLE ROLE OF THE HUMAN NACETYLTRANSFERASE 2 GENE POLYMORPHISMS IN THE DEVELOPMENT OF ISONIAZID RESISTANCE
98
Introduction
101
Materials and Methods
104
Results
108
Discussion
111
CHAPTER 5: SOCIO-ECONOMIC FACTORS WHICH MAY PLAY A ROLE IN THE DEVELOPMENT OF ISONIAZID RESISTANCE
121
Introduction
124
Materials and Methods
127
Results
132
Discussion
140
CHAPTER 6: COMPREHENSIVE METHODOLOGY
150
Methods 1.
Study Site
151
2.
Collection of Cultures
151
3.
M. tuberculosis DNA
152
4.
Cluster Analysis
153
5.
Collection of Blood
154
6.
Human DNA Extraction
154
7.
Cleaning of Contaminated Human DNA Samples
156
8.
Quantification of Human DNA
156
9.
Oligonucleotide Primers and Probes
157
10.
DNA Repair Gene Primers
157
11.
DNA Repair Gene Probes
158
12.
NAT2 Primers
160
13.
SNaPshot Primers
160
14.
Polymerase Chain Reaction (PCR)
161
15.
PCR Conditions
161
16.
Agarose Gel Electrophoresis
163
17.
PCR Dot-Blot Hybridization with Allele Specific Probes
163
18.
Blast Similarity Searches
165
19.
SNaPshot Primer Extension Genotyping Method
166
20.
N-Acetyltransferase Genotyping
167
21.
RFLP / AS-PCR Analysis
168
22.
Sequencing
169
23.
Clinical and Socio-Demographic Data Collection
170
24.
Statistical Analysis
171
Materials 1.
Buffers and Solutions
173
CHAPTER 7: CONCLUDING REMARKS
177
REFERENCES
183
APPENDICI
1
RISK FACTORS ASSOCIATED WITH ISONIAZID RESISTANCE IN TUBERCULOSIS
CHAPTER 1
LITERATURE REVIEW
2
INTRODUCTION
HISTORY OF TUBERCULOSIS
Mycobacterium Tuberculosis (M. tuberculosis) is the causative agent of Tuberculosis (TB) (163), an infectious disease which predominantly infects the human pulmonary tissue.
It was postulated that TB originated within the human race, via possible
transmission of the disease through Mycobacterium bovis in ancient times, as skeletal remains demonstrate pathology consistent with TB infection (34). In addition to the anatomical evidence, deoxyribonucleic acid (DNA) from ancient remains was amplified with Mycobacterium specific primers and generated positive results. Sequence analysis showed that the DNA was homologous to that of M. tuberculosis (112). However, current sequence-based analysis states that M. tuberculosis and Mycobacterium bovis arose from a common ancestor, with M. tuberculosis the first to arise (183). In ancient Greece, Hippocrates spoke of “phthisis” and noted that the disease was very common and always fatal.
However, TB was not considered as a major threat to health until the industrial revolution in 1600 A.D. (148). In 1882, Robert Koch identified the etiology of TB and what was to become one of humanity’s deadliest enemies. In 1853, 29 years prior to Koch’s discovery, Verdi wrote one of his most popular operas; La Traviata. It was the first opera in which a person dies from consumption and it highlighted the shocking immediacy of the disease in its own time. TB, however, is not particularly selective with regards to the
3 socio-economic status of its human host, since people such as Anna Pavlova, Robert Louis Stevenson, Mozart and Napoleon, to name but a few, were victims of TB. None the less TB is strongly associated with poverty and poor socio-economic circumstances.
IMPACT OF TB
It was in the 1980’s that the dilemma of modern TB management began with the emergence of multi-drug resistant TB (MDR-TB). That decade also saw the start of the AIDS (Acquired Immune Deficiency Syndrome) pandemic.
TB poses a substantial threat to civil health and was declared a global emergency by the World Health Organisation (WHO) in 1993 (130). This state of emergency was due to the fact that, prior to the 1980’s, the incidence of TB was declining and it was now realised that the incidence was in reality on the increase (1). Every year there are approximately 8-10 million new reports of people diseased with TB worldwide, which leads to 3 million deaths per annum, about five deaths every minute (126). Of those infected, approximately 10% will develop active TB (178). It is estimated that between 2002 and 2020, nearly 1000 million people will be newly infected, over 150 million people will become diseased and 36 million will die of TB (175) (Figure 1).
In
Southern Africa, the TB epidemic has developed into one of the worlds most severe (8). In 2000, the incidence of TB in adults in the Boland region of SA was approximately 1350 per 100 000 population, with an incidence of drug resistance of 24 per 100 000 population and multidrug resistance of 10 per 100 000 population (8,10).
4
Figure 1. The estimated global TB incidence rates in 2002 (175).
5 TB CONTROL
The lack of effective control of TB prompted the WHO to devise a strategy that could be easily implemented in health intervention policies as a safeguard against the rising TB epidemic and subsequent acquisition of drug resistance. This strategy is called “Directly Observed Treatment Short course” (DOTS) and relies on the following ground rules: (178) 1. government obligation, 2. early case detection using sputum smear microscopy among individuals seeking medical attention for prolonged cough, 3. standardised short-course chemotherapy under appropriate case-management conditions, including directly observed treatment, 4. habitual, constant drug supply and, 5. a standardised recording and reporting system that permits assessment of individual patients as well as overall programme achievement.
The ultimate goal with the DOTS strategy is to minimise transmission of M. tuberculosis and to effectively cure every individual patient with the prescribed first-line drugs: Isoniazid (INH), Rifampicin (RIF), Pyrazinamide (PZA), Ethambuthol (EMB) and Streptomycin (STR) (178). This goal may be realised since DOTS is inexpensive, has a cure rate of up to 95% and prevents new infections by curing infectious patients. It also minimises the development of MDR-TB by ensuring that the patient stays compliant for the duration of the full course (116). However, the DOTS programme for certain low-
6 income countries offers no priority to effectively treat individual patients with MDR-TB and subsequently the initial treatment of MDR-TB is delayed, leaving patients infectious for a longer period of time (157).
The increase and spread of MDR-TB in patients, as well as HIV co-infection in TB patients, complicate the DOTS strategy and thus a new strategy was developed to combat these incriminating factors (178). This DOTS-plus strategy, only implemented where DOTS are effectively managed, is an expansion of the DOTS programme and focuses on the treatment of patients with known or suspected drug resistance, with second line drugs (178). The DOTS / DOTS-plus programme allows for the identification of possible risk factors since the recording and reporting of patient information is crucial in the control of TB. Furthermore, this programme, adopted in 1996, is implemented in all provinces of South Africa and subsequent analysis of patient records might be insightful with regards to the emergence of MDR-TB in South Africa. Nonetheless, the inefficiency of both health care and TB education programmes currently result in ineffective treatment of TB.
DRUG RESISTANCE
STR, an aminoglycoside, was administered for the first time in 1944 (121), but as with most bacterial infections, resistance against STR soon occurred and the administration of two or more drugs proved to be more effective. An even greater advance against TB came when INH (isonicotinyl hydrazine) was introduced. It was first synthesised in 1912
7 but its therapeutic effect against tuberculosis in humans was only detected in 1952 (134). These discoveries started the era of modern chemotherapy.
Within approximately ten years after the introduction of INH, cycloserine (CS), PZA, EMB, and RIF were introduced as anti-TB drugs. INH remains the most important single agent for the treatment of tuberculosis (72). Globally, TB control efforts are seriously threatened by the increasing rate of drug resistance (50,117,138,177). The emergence of drug-resistant strains of M. tuberculosis, especially MDR strains, defined as resistant to at least INH and RIF, poses a threat to the success of TB control programmes. Drug surveillance studies by the WHO in 64 countries indicated that 3.4% of TB cases are MDR and that there were an estimated 237 000 new cases of MDR-TB world-wide in the year 2000 (46).
Acquired drug resistant strains arise when treatment is intermittent or otherwise inadequate and primary resistance occurs when a patient is infected with a resistant strain. Furthermore, the rising HIV epidemic in many countries, especially in sub-Saharan Africa where the prevalence of TB infection is high, further complicates the control of drug susceptible and resistant TB (73).
Approximately 98% of all TB deaths occur in
developing countries where there are limited resources and surveillance for drug resistance is irregular (130). Several factors may influence the measure of success of treatment programs. The main factors responsible for the increase in the global TB burden are poverty, poor programme management (inadequate case detection, diagnosis
8 or cure), and population increase (8). Outbreaks of MDR-TB in institutions such as hospitals (20,32,47,57) and prisons (26), and among health care workers (18,84,120) in the developed world have focused attention on MDR-TB as a major health issue.
The most widespread MDR-TB outbreak reported to date occurred in 267 patients from New York, who were infected by the Beijing/W strain (62). These strains were resistant to all first-line anti-TB drugs, resulting in prolonged infectiousness and exposing other individuals at risk of nosocomial infection with these difficult to treat strains. Since then, the Beijing/W genotype family of M. tuberculosis has been the focus of extensive investigation and has been found to be widely spread throughout the world (65), including South Africa (157). It also constitutes the major family of MDR-TB isolates currently circulating in Russia (106). MDR outbreaks are not restricted to the Beijing genotype since isolates with different characteristics have also been reported in less significant outbreaks of MDR-TB (89,123). An alarmingly high concentration of this particular family of strains was found in the Beijing area of China, where more than 85% of all isolates belonged to this family. The Beijing strains are strongly associated with drug resistance in other countries such as Russia, Vietnam and Estonia (45,65,90).
The countries with the highest case load of TB in the world, in decreasing order, are : India, China, Indonesia, Bangladesh, Nigeria, Pakistan, South Africa, The Philippines, Russia and Ethiopia (178).
9
In developed countries, immigrants and HIV-positive individuals are more likely to have drug resistant TB (41,48,54,67,79,103). However, reports from developing countries concerned with the characterisation of dominant drug resistant strains are limited due to the fact that molecular fingerprinting is not done in routine surveillance (133,157,170). In South Africa, MDR-TB was first identified in 1985 in the Western Cape Province and the incidence thereof increased to around 2% (172). Currently there are more than 3000 individuals newly infected with MDR-TB in SA per annum (178).
DRUG SUSCEPTIBILITY TESTING Effective management of TB relies on accurate diagnosis. Culture and drug sensitivity testing (DST) on M. tuberculosis is an expensive and slow diagnostic technique, which costs about 70 ZAR (~10.62 USD) per sensitivity test and takes on average 41 days to establish the resistance phenotype.
The period of time taken to determine drug
susceptibility by initially growing the specimen on culture (3 weeks) as well as the ensuing DST (3 to 6 weeks), is approximately 6 – 9 weeks. These drawn out procedures can contribute to the transmission of MDR-TB (163) since initiating treatment during this crucial period is delayed. This is especially true in lower income countries where DST facilities are not part of routine diagnostic procedures and where current drug-sensitive strains may develop resistance over time (156,163).
10 Identification of MDR strains can be established by means of DST. Thus, after an initial positive culture is obtained, DST is done on a subsequent subculture. DST is done by conventional methods (absolute concentration method, the resistance ratio (RR) method and the proportional method) or by means of radiometric methods such as the BACTEC method.
The proportion method is frequently done to determine drug susceptibility of
M. tuberculosis. The results obtained from this method are reported as the percentage of the total bacterial population resistant to a specific drug, which is defined as the amount of growth on a drug-containing medium compared with growth on a drug-free control medium (38).
Each drug is tested at its critical concentration, defined as the
concentration that inhibits growth of the majority of wild-type (susceptible) strains of M. tuberculosis without noticeably affecting the growth of resistant mutants present. Critical concentrations for the front-line drugs for different susceptibility testing methods are listed in Table 1. Table 1. Critical drug concentrations for routine susceptibility testing (mg/ml). Adapted from the Department of Health (38). Radiometric
Conventional
(15 days)
(3 to 6 weeks)
Drug Bactec 12B
Middlebrook 7H10
Löwenstein-Jensen
Isoniazid
0.1
0.2
0.2
Rifampicin
2.0
1.0
40.0
Ethambutol
2.5
5.0
2.0
Streptomycin
2.0
2.0
4..0
11 When 1% or more of the bacterial population become resistant to the critical concentration of a particular drug, the M. tuberculosis isolate is considered as exhibiting resistance toward that drug.
MOLECULAR EPIDEMIOLOGY
Modern TB epidemiology combines molecular techniques (to trace specific strains of M. tuberculosis) with conventional epidemiologic methodology, to understand the distribution of TB in populations (143,155,167) and employs genetic markers as targets for molecular classification. The genome of M. tuberculosis is highly conserved, and contains polymorphic regions associated with insertion sequences (IS), as well as repetitive elements (19,71). These are used as probes in DNA fingerprinting of M. tuberculosis, and rely on differentiating between the IS6110 copy numbers of each strain. The most widely used marker applied in DNA fingerprinting is the transposable element IS6110, which varies in both copy number and location in the genome (110,158,167).
The IS6110 insertion element is found in most M. tuberculosis isolates and because transposition is believed to happen in an unsystematic fashion, a huge range of different insertion patterns can be distinguished on the chromosomes of different clinical isolates (169). IS6110 is normally considered to be variable enough to differentiate between unrelated strains but stable enough to remain consistent in related strains (110). Isolates from patients infected with epidemiologically unrelated strains (unique isolates) have
12 different RFLP patterns, whereas those from patients with epidemiologically linked strains usually have identical RFLP patterns (16).
Strains that recently derived from a common ancestor have the same DNA fingerprint, and these strains can be classified into epidemiologically linked clusters (158). Clustering is indicative of recent transmission or a common source of infection (16,71).
The
investigation of the clinical, social, demographical and microbiological factors associated with clustered cases is of utmost importance to identify risk factors which might contribute to the transmission of TB (71).
The use of IS6110 to distinguish between different families of M. tuberculosis is very sensitive but time-consuming and a quicker and easier PCR-based technique to type different strains of M. tuberculosis was subsequently developed, based on the direct repeat (DR) locus (158). The DR locus in M. tuberculosis contains copies of a 36-bp direct-repeat, which are separated from one another by spacers that have different sequences.
These spacers are highly conserved among different strains of M.
tuberculosis, and all strains have the same overall arrangement of 43 spacers which appear in different distinguishable patterns due to the absence of different combinations of these spacers.
Strain differentiation is thus determined by the absence or presence of specific spacers and the different strain families, characterised by the distinct “signature” of each isolate, are designated spoligotype reference numbers (168,169). These spacer oligonucleotide
13 patterns (spoligotyping patterns), increase the power of strain identification (16), and thus DNA fingerprinting of M. tuberculosis makes it possible to trace the transmission of isolates within a community (167). numbers of
Other strain typing methods include: Variable
tandem repeats (VNTRs), Mycobacterial interspersed repetitive units
(MIRUs) and fluorescent amplified-fragment length polymorphisms (109,158).
The
application of IS6110 fingerprinting and spoligotyping led to the finding that the Beijing/W family predominates in local communities. This predominance suggests that this strain might have a selective advantage during recent transmission compared to other M. tuberculosis genotypes (149,158).
DEFINITION OF THE BEIJING/W STRAIN OF M. TUBERCULOSIS
The genetically highly conserved Beijing family of M. tuberculosis (resistant and susceptible strains) was first described in 1995 (88). They share an identical high copy multi-banded IS6110 RFLP banding pattern and closely related spoligotype patterns. The drug-resistant Beijing strains, all belonging to Principal Genetic Group 1, are further characterised by the absence of resistance conferring gene mutations and polymorphisms in the katG 463 codon and gyrA 95 codon , characteristic of all Group 1 strains (19,20). These mutations and polymorphisms however, are considered as wild type sequences since Group 1 strains are evolutionary older than those of Groups 2 and 3.
In North America, a multidrug-resistant strain of M. tuberculosis (the W strain) emerged and it was shown that the IS6110 RFLP and spoligotype patterns of the W strains had a
14 high degree of similarity to that of the Beijing strains (88).
Subsequent molecular
genotyping confirmed that the MDR-TB strain W family, responsible for a large MDRTB outbreak in New York in the 1990s, is a member of the Beijing genotype (62). These two strains, which independently originated in different countries, represent the same genotype and are now referred to as the Beijing/W family.
However, a more recent group of isolates have been identified which belong to the Beijing/W family. This group is referred to as Atypical (or Ancestral) Beijings, since their IS6110 RFLP profiles are similar but not equally identical (158). The similar RFLP patterns are indicative of being distantly related to the Beijing/W family (107), which is an indication that the atypical Beijings diverged earlier in the evolution of M. tuberculosis (88). Furthermore, it is suggested that the Beijing/W genotype may have a proclivity for acquiring drug resistance since it is widely associated with outbreaks of multidrug-resistant TB (65).
In a recent study, it was reported that the Beijing/W strain exhibits mutations in putative mutator genes, which may give clues as to understanding why the Beijing/W strain of M. tuberculosis has an increased adaptability to adverse conditions such as stress from both exposure to exogenous antituberculosis drugs as well as endogenous attack from the harsh intracellular environment (125).
15
MOLECULAR MECHANISM OF ISONIAZID RESISTANCE INH enters the mycobacterial cells by means of passive diffusion across the mycobacterial cell envelope (83), a superpolymer of covalently attached subunits (142). These covalent structures terminate in a lipophilic layer of extraordinarily long-chain αbranched β-hydroxylated fatty acids, which are called the mycolic acids.
The
biosynthesis of mycolic acids is essential for cell structure and once the cellular integrity is disrupted, the bacterium dies (17,142).
PERMEABILITY OF THE CELL WALL AND ISONIAZID RESISTANCE
The basic innate drug resistance mechanism in mycobacteria is that of drug efflux and this contributes to natural or acquired resistance (94). Efflux pumps are proteins that transport antibiotics to the outside of the bacterium, generating low levels of resistance to antibiotics and have been identified in all bacteria (166).
Mycobacteria possess innate
resistance to most antibiotics as a consequence of the gradual uptake of drugs across the hydrophobic cell wall envelope. It is believed that considerable contributions to acquiring resistance are made from efflux transport proteins, such as the efflux protein efpA, by reducing the cytoplasmic drug concentration to sub-inhibitory levels (165). There is however no evidence of strain dependence for efflux proteins since it is common in all M. tuberculosis strains.
16
INH is further neutralised within M. tuberculosis by the up-regulation of arylamine Nacetyltransferase, by means of inhibiting NAD+-binding proteins (166), as well as the upregulation of enzymes which act as antioxidants, when compensating for the loss of the catalase-peroxidase protein. Isolates of M. smegmatis deficient in the energy-dependent NADH dehydrogenase efflux pump exhibit resistance to INH (29) and it has been shown that the combination of the absence of mycolic acids with the stabilising consequence of glycopeptidolipids confers innate resistance to M. avium (29,100).
During ineffective TB management and non-compliance, it has been shown that overexpression of existing efflux pumps in reaction to prolonged exposure to sub-effective concentrations of antimycobacterials, lead to increasing levels of resistance to the drugs used in chemotherapy (165). The accumulation of INH in M. smegmatis is known to be modulated by active extrusion systems (29). Furthermore, the role of efflux pumps in acquiring drug resistance might be elaborated by the recent discovery that INH-sensitive M. tuberculosis isolates, possess a reserpine-sensitive efflux mechanism (reserpine being a pump inhibitor), and that this is induced by step-wise exposure to INH, generating highlevel resistance to INH (166).
One particular gene which is implicated in INH resistance, and hence drug tolerance, is iniA.
Very recently it was found that a deletion in iniA results in an increased
susceptibility to INH (30) and thus the role of iniA was assigned as being part of an efflux pump. However, the authors could not show that iniA directly transports INH from the
17 cell.
In addition, it was found that the whiB7 gene (which confers resistance to
antibiotics having specific targets) in mycobacteria may act synergistically with the cell wall to provide high levels of innate resistance (108).
The genome of the M. tuberculosis laboratory strain H37Rv has 20 open reading frames which code for putative efflux proteins (31) and the permeability barrier, imposed by the mycobacterial cell wall, contributes to the development of low-level drug resistance (129). The effectiveness of a pump is related to its number of units and a possible reason for the observation of differences in susceptibility might be due to an increased number of efflux pumps as well as the fact that different isolates from the same strain exhibit different levels of susceptibility to antibiotics (132,166).
DRUG RESISTANCE CONFERRING MUTATIONS
Strains develop drug resistance by means of mutations in genes of which the gene products either activate the drug or in turn are the drug target (145). However, while many mutations in a number of different genes have been found to be associated with INH resistance, about 25-30% of INH resistant clinical isolates lack a known INH resistance gene mutation (163).
Several mechanisms towards resistance against anti-tubercular drugs are employed by M. tuberculosis. Presently, a total of 12 loci responsible for drug-resistance to first-line drugs in M. tuberculosis have been identified (164), and six of these (katG, inhA, aphC,
18 kasA, ndh and oxyR-ahpC) are associated with resistance to INH.
These resistant
associated point mutations, deletions or insertions and their targets implicated in resistance to anti-mycobacterial drugs are summarised in Table 2.
19 Table 2. Cellular targets for anti-mycobacterial drugs and gene mutations responsible for resistance in M. tuberculosis. (128,163). Drug
Cellular
Gene locus
Target enzyme/functional role
katG
Catalase-peroxidase/activation of prodrug
inhA
Enoyl reductase/mycolic acid biosynthesis.
ahpC
Alkylhydro-peroxidase
kasA
β-ketoacyl synthase/mycolic acid biosynthesis
ndh
NADH dehydrogenase/regulation of NADH/NAD+
oxyR-ahpC
detoxifying enzymes/regulatory protein
target Isoniazid
Cell wall
Rifampicin
Nucleic acids
rpoB
Β subunit of RNA polymerase/transcription
Streptomycin
Protein synthesis
rpsL
Ribosomal protein rRNA/translation
S12/translation
rrs Ethambutol
Cell wall
Pyrazinamide
Unknown pncA
Fluoroquinolones Nucleic acids
embB
gyrA
Arabinosyl transferase/arabinan polymerization.
Pyrazinamidase/activation of prodrug. DNA gyrase subunit/DNA replication
16S
20 THE CATALASE-PEROXIDASE ENZYME AND INH RESISTANCE
INH is a prodrug which needs to be activated by the mycobacterial katG gene product, catalase peroxidase, by producing a reactive antimicrobial intermediate. The physiological role of KatG is that of protection, by resisting the low pH found within the human macrophages during the oxidative burst where free oxygen radicals are converted to peroxides (H2O2). KatG activity counteracts this harmful reaction in the following process: (83) 2H2O2 Æ 2H2O + O2 It thereby protects the bacilli from oxidative peroxidation.
INH resistance in M. tuberculosis may also be due to the loss of catalase-peroxidase activity within the mycobacterial genome (102,181). Genetic studies revealed that when a functional katG gene was introduced into INH resistant strains of M. smegmatis and M. tuberculosis, deficient of catalase-peroxidase, these strains were capable of restoring their susceptibility to INH and thus katG deletions are key to understanding INH resistance (180,181).
However, the complete deletion of the katG gene only occurs in a small
fraction of highly INH resistant strains (80).
MUTATIONS WITHIN THE KATG GENE
The genome of M. tuberculosis possesses repetitive DNA sequences, and the region within which katG lies, exhibits relative instability (142,182). The instability within the
21 katG region may contribute to the relatively elevated rates of INH-resistant mutants, which are approximately 1 in 105 – 1 in 106 organisms generated during every in vitro selection of INH (100,142). Furthermore, potential recombination hotspots are loci of substantial interstrain variation (159,161) which may further account for mono- or multidrug-resistance to INH.
Within a microbial population, genomic material may not be fixed, as differences in selective forces such as exogenous exposure to antimicrobials and other DNA damaging agents favour distinctive phenotypes (13). A model organism for studying genome plasticity is that of Helicobacter pylori. H. pylori, like M. tuberculosis, has extensive, non-randomly distributed repetitive DNA sequences (13) and both pathogens are well adapted to their particular niches. Recombination among identical repeats in H. pylori contribute to the variation within individual hosts since it is speculated that this organism might have evolved mechanisms to facilitate such plasticity (13).
The mutations
involved are located either on the N-terminal or C-terminal of the katG gene product (80).
The N-terminal of the katG protein is associated with the active site of the catalaseperoxidase enzyme and most of the mutations conferring a high level of INH resistance are located between codons 138 and 350. The most frequent mutation however, is a point mutation at codon 315 of the katG gene (142) which results in an amino acid substitution by replacing serine with threonine (Ser315Thr). The most frequent polymorphism associated with the katG 315 mutation is that of the katG 463 codon where the Arginine
22 residue is replaced by Leucine. This codon is situated at the C-terminal of the katG gene and is not readily associated with the catalase-peroxidase active site, and hence it is considered as a natural polymorphism (80,163).
It is estimated that the katG 315 mutation and katG 463 polymorphism occur in 55 – 75% of all INH resistant isolates (111,127,142,163) and are characteristic of the Beijing/W family (127). The majority of mutations in clinical isolates are missense mutations, and this indicates that even though the activity of KatG is reduced in vivo, the reduced activity still confers a selective advantage since nonsense mutations results in truncated proteins (83).
INCREASED EXPRESSION OF TARGET PROTEINS The loss of catalase-peroxidase is linked to the increased expression of compensatory M. tuberculosis genes such as ahpC, InhA, KasA and AcpM (80,142). Mutations can occur at several sites in the promoter regions of the genes which result in altered transcriptional activity (80,142). Mutations in the coding regions of these genes, however, are less frequently observed.
Nonetheless, mutations in these promoter sequences do not
completely elucidate INH resistance in clinical isolates and recently it has been found that mutations in the ndh gene, which encode for NADH dehydrogenase, may be an additional marker for INH resistance in M. tuberculosis (91). implicated in INH resistance will be discussed briefly:
The gene products
23 THE ENOYL REDUCTASE ENZYME AND INH RESISTANCE
After initial activation of INH by catalase-peroxidase, an ensuing target is the Enoyl acyl carrier protein (ACP) reductase, encoded by the inhA gene (128,176). The activated INH derivative binds to the inhA-NADH complex, forming a ternary complex, which results in INH resistance due to inhibition of mycolic acid biosynthesis. Point mutations in the structural inhA gene result in amino acid substitutions, resulting in the decreased affinity of inhA for NADH, and presently only six mutations have been reported on. However, mutations in the promoter region of this gene cause the over-expression of inhA, with the resulting effect of low levels of INH resistance (127,142).
THE ALKYLHYDRO-PEROXIDASE ENZYME AND INH RESISTANCE
Alkylhydro-peroxidase, encoded by the ahpC gene, functions as a detoxifying agent and has its effects on organic peroxides (139). Promoter mutations in this gene in INH resistant isolates also result in its over-expression and function as a compensatory mechanism for the loss of catalase-peroxidase activity due to mutations in the katG gene (128). High levels of peroxides within the cell result in the over-expression of ahpC to combat oxidative damage. However, the increased expression does not permit any effect on INH and results in the emergence of INH resistance (39,142).
24
THE Β-KETOACYL SYNTHASE ENZYME AND INH RESISTANCE
In addition to the Enoyl acyl carrier protein (ACP) reductase, another target after initial activation of INH is β-ketoacyl ACP synthase (KasA), encoded by kasA (128,142). This enzyme is also involved in the mycolic acid biosynthesis and low levels of INH resistance have also been attributed to mutations in this gene (99,128).
THE NADH DEHYDROGENASE ENZYME AND INH RESISTANCE
NADH dehydrogenase, an essential respiratory chain enzyme, is responsible for the regulation of NADH/NAD+ in the cell (128). It is encoded by ndh and it has been found that missense mutation in this gene results in INH resistance due to reduced enzymatic activity. The increased NADH accumulation and decreased NAD depletion result in the inhibition of the biosynthetic pathway which is crucial for metabolic flux (91).
THE OxyR ENZYME AND INH RESISTANCE
The OxyR enzyme, encoded by oxyR, controls the expression of katG and ahpC during times of oxidative stress. It is a regulatory protein that functions as both an oxidativestress sensor and activator of gene transcription (146). Mutations in this intergenic region lead to impaired gene transcription of katG and ahpC and result in INH resistance (128).
25
POSSIBLE ROLE OF DNA REPAIR IN THE DEVELOPMENT OF DRUG RESISTANCE
The extent of damage to DNA is measured by the DNA repair rate. The major response to DNA damage is by means of induction of genes which are important for DNA repair and cell division (24).
Mutator phenotypes/genotypes are direct consequences of DNA repair and strong selective pressure for systems that are capable of preserving genetic information is seen in all genomes (14). However, as essential as DNA repair might be, “evolvability” (the ability to create a certain amount of uncorrected mutations) is selected for as well in the progression of natural evolution. The necessity for high fidelity DNA repair as well as the necessity for evolvability is two-fold, and it is this dichotomy that defines the organisation of the DNA repair systems (14). The bacterial genome of E.coli has been extensively used as a model system in studies regarding DNA repair genes in eukaryotic organisms. In addition to proofreading by DNA polymerases, there are a variety of bacterial systems implicated in repair of mutations and DNA damage and the proteins involved can be grouped into the following major functional categories: (5,14,105,124)
(i)
direct damage reversal (DDR)
(ii)
base excision repair (BER)
(iii)
nucleotide excision repair (NER)
(iv)
mismatch repair (MMR),
26 (v)
recombinational repair (RER), and
(vi)
SOS repair and mutagenesis.
The bacterial genome encodes for around 115 proteins involved in bacterial DNA repair, and this metabolically conserved state is maintained throughout evolution (14).
DNA REPAIR IN M. TUBERCULOSIS
In a recent systematic review of the worldwide occurrence of Beijing/W strains, concern was raised that these strains may be disseminating and might have a predisposition for acquiring drug resistance, due to their ability to mutate more rapidly than other strains (65). When macrophages are activated by the presence of tubercle bacilli, they produce reactive oxygen intermediates (ROI) such as peroxide, and reactive nitrogen intermediates (RNI) such as nitric oxide, which damage the bacterial DNA (24,59) through oxidation and alkylation.
In addition to this host immune response, bacterial DNA damage also results from external factors such as the administration of anti-tubercular drugs (136), which is mostly directed against replicating bacteria. However, outbreaks of MDR-TB are associated with the emergence of the bacteria from the dormant (stationary) phase, and this stationary phase might be crucial in explaining why 25-30% of INH resistant clinical isolates lack a known INH resistance gene mutation.
27 During hostile environmental conditions, bacterial growth of E.coli is arrested, which causes particular alterations in the cell-envelope lipid-fatty acid structure, which is needed by the bacillus to survive (40). It is suggested that the membrane fatty acids serve as endogenous reserves which provide energy for maintaining cell integrity. The same mechanism might be extrapolated to M. tuberculosis. In addition to alkylation and oxidation, as well as exposure to anti-TB drugs, bacterial DNA damage to the cell wall, might be mediated by the bacilli themselves, in order to sustain the dormant bacteria. However, dormant non-growing cells are able to evolve (55) and DNA repair systems are important for the successful emergence from the dormant state (105).
With the completion of the genome sequence of the laboratory strain of M. tuberculosis, H37Rv, it was found that it consists of 4 411 529 base-pairs (bp) which can potentially encode 3 924 genes (31,122). A recent in silico study tried to identify homologues of TB DNA repair genes (mut genes) in E.coli and other organisms (105). The results showed that M. tuberculosis lacked genes involved in the highly conserved methyl-directed mismatch DNA repair system, such as mutS, mutL, or mutH, which makes it unique and thus differentiating it from E.coli. Nonetheless, in vitro it was found that bacteria which possess MMR systems had the same spontaneous mutation frequency (35,125).
The
genes identified within the genome sequence of M. tuberculosis revealed that the repair systems can be categorised into three major functional repair categories: (105)
(i)
excision repair (base and nucleotide) and direct damage reversal
28 (ii)
recombination repair, and
(iii)
SOS repair and mutagenesis.
M. tuberculosis is deficient of MMR, as mentioned earlier, commonly found in other bacteria. Some of the major genes that were found to be involved in the abovementioned categories are summarised in Table 3. For the purpose of this thesis, only BER as well as SOS repair and mutagenesis will be elaborated on.
29 Table 3. Homologues of E. coli genes found within M. tuberculosis, involved in DNA repair. (I) excision repair and direct damage reversal, (II) recombination repair and (III) SOS repair and mutagenesis. Adapted from Mizrahi (105).
Repair Category GENE
I
ogt
9
Damage Reversal
mutT (mutT2 and Rv3908)
9
Removal of damaged nucleotide
recA
9
lexA
9
polA
9
9
Post incision events
ssb
9
9
Protein binding of single-stranded DNA
uvrA
9
II
9
III
Function
9
Regulation of NER
9
Regulation of NER
9
Excision repair
dinP
9
Polymerase IV
dinG
9
helicase
recN
9
9
recombination
ruvA
9
9
resolution
30 BASE EXCISION REPAIR
The BER system is a multiple enzymatic DNA repair mechanism in which each enzyme works separately, and has been maintained throughout evolution. It corrects damage caused by hydrolysis and exposure to ROI and RNI that oxidize and alkylate DNA (105,137). The BER pathway protects DNA from a variety of other damaging agents, such as: uracil, hydroxymethyluracil, methylcytosine, G-T mispairs, 3-methyladenine, 7methylguanine, 3-methylguanine, 8-hydroxyguanine, and pyrimidine dimers (61). The BER system is therefore responsible for the elimination and subsequent replacement of deaminated, oxidized or alkylated bases (5).
SOS REPAIR AND MUTAGENESIS
The majority of the genes listed in Table 1.3 are part of the SOS response.
M.
tuberculosis possesses a functional SOS system, which is characterised by regulatory elements. The SOS response is regulated by the repressor protein LexA in combination with RecA, involved in recombinational repair, which acts an activator. The expression of recA, an essential component in DNA repair and recombination, is induced by DNA damage in M. tuberculosis which binds to single-stranded DNA (24,105). However, nontargeted SOS-induced mutations (not associated with mutagenic treatment) stimulated AT to G-C transversions (104).
Environmental factors, such as anti-tuberculosis agents,
could be attributable for the induction of SOS response which increases the rate of spontaneous mutagenesis (131).
31
ADAPTIVE MUTAGENESIS
Spontaneous mutations not only occur in growing bacteria, but in dormant cells under selection for a specific phenotype as well.
This phenomenon is primarily due to
exposure to non-lethal selection and is referred to as adaptive mutation or both starvationassociated and stationary-phase mutations (105,114).
Further studies in E.coli showed
that repair-defective mutator activity may be beneficial under conditions of stress (105).
In mammalian species, cancer cells are mutators. Tumorogenesis is a form of evolution which is under constant immune surveillance and is kept in control by tumour suppressor genes such as p53. However, mutations in these genes allow for the expression of tumours. The somatic cell population consists of individual cells within the tumour, and is classified as either being benign or malignant. The malignant forms grow faster and are considered to be “mutator phenotypes” (87). However, since normal non-dividing somatic cells exhibits low mutation rates, the increased mutations in tumour cells are manifestations of mutator phenotypes which are present in the early stages of tumorogenesis. Thus, the co-operative action between increased mutagenesis and clonal selection offers a mechanism for the selection of cells with increased proliferative advantage (96).
Although such an increased advantage is deleterious to humans, an increased spontaneous mutation rate might be beneficial in M. tuberculosis. Adaptive mutation is the result of a
32 variety of responses in which mutations in organisms are formed, after being exposed to growth-limiting environments which produce genetic rearrangements, and allows for the fast emergence from the dormant state (75). The Beijing/W family may have a mechanism for recombination-dependent adaptive point mutations. Mutations in such a system occur from DNA replication established by homologous recombinational repair of double stranded breaks (DSB) in the stressed, dormant bacilli (74,75).
The DNA synthesis is believed to occur in environments where the SOS response is induced and MMR is absent, which leads to the persistence of replication errors. Thus, the distinguishing characteristics of this point mutation mechanism includes the requirement for DSBs, DSB-repair proteins, the SOS response and its error-prone pol IV, absence of MMR, and most importantly its association with global hypermutation in a small sub-population (75). It was found that in the Lac system of E.coli, Lac+ mutants had an increased frequency of unrelated mutations, in contrast to Lac- cells starved on the same plate indicating that adaptive point mutations appeared in a sub-set of the population that underwent global hypermutation (66).
Furthermore, mutations in
stationary-phase E.coli cells indicated that hypermutation in the chromosome is promoted by recombination (25).
HYPERMUTATORS
Mutation is the ultimate source of heritable variation on which natural selection acts. One evolutionary theory postulates that a mutation rate evolves as low as possible, since
33 most de novo mutations are either deleterious or silent. In contrast, favourable mutations, specifically those conferring antibiotic resistance, are rare (21,44,92). However, it has been shown that clones which exhibit an elevated mutation rate (hypermutatable strains or mutators) can increase in frequency in in vitro bacterial populations (33,63). Furthermore, it has been shown that in M. tuberculosis, resistance to antimicrobial drugs are the consequence of spontaneous mutations (favourable rare mutations in this case), in genes that either target the antibiotic agent, or enzymes that are involved in activation of the pro-drug (145).
Mutator alleles may play an important role in adaptive evolution, as suggested by in silico simulations, since these alleles can accelerate the evolutionary rate under some conditions and increase the possibility of favourable mutations (21). It is proposed that these mutators “hitchhike” with the favourable mutations they have originated (151) and thereby become fixed within the population. In this way, the acquisition of a mutator phenotype increases the chances of acquiring antibiotic resistance by mutational events (21), since one percent of pathogenic strains are hypermutators (98).
Arbitrary hypermutation in particular genes is an evolutionary method by which many pathogenic micro-organisms defend themselves against their host (22), thereby indicating that a high mutation rate may play an important role in adaptive evolution.
34 SELECTION FOR MUTATOR PHENOTYPES
Drug induced mutator phenotypes have been observed in other bacteria and it has been suggested that antibiotics may select for mutator phenotypes (15,64). INH inhibits the biosynthesis of cell wall mycolic acids and it was found that E.coli cells which were exposed to translational inhibitors displayed mutator phenotypes (131). It is therefore possible that INH treatment may give a bacterium with a mutator phenotype, a selective advantage (with or without induction of genomic alterations in the bacterium).
It is therefore possible to speculate that the Beijing INH mono-resistant isolates constitute an emerging strain, which by further selection, may develop additional drug resistance to ultimately become the full Beijing/W MDR-TB strain over time.
Single Nucleotide Polymorphisms (SNPs) are less subject to selective pressure (217) than other distinguishing characteristics such as chromosomal mutations in genes which confer resistance to antituberculosis drugs. In a recent study it was found that several SNPs in numerous genes which are either involved in mycolic acid biosynthesis or upregulated as a reaction to cellular accumulation of INH were exclusively linked to INH resistant clinical isolates (128). The recent completion of the genome sequence of two different M. tuberculosis strains, the laboratory strain H37Rv (Principal Genetic Group 3) (219), and the clinical strain CDC1551 (Principal Genetic Group 2) (220), as well the preliminary sequence data for the Beijing/W strain, M. tuberculosis 210 (Principal Genetic Group 1) (6,128) allows for genome comparisons between these three
35 mycobacterial groups. Synonymous SNPs in M. tuberculosis have been revealed by comparative sequence analysis between these and are useful for evolutionary, pathogenic, and epidemiological studies (11,58).
POSSIBLE ROLE OF THE HUMAN N-ACETYLTRANSFERASE 2 GENE IN THE DEVELOPMENT OF ISONIAZID RESISTANCE
It was observed that urinary excretion of unchanged INH, after repeated administration, was constant in any individual, but the inter-individual excretion was clearly different for each individual (101). It was later found that this phenomenon was due to differences in the individual’s ability to metabolise INH to acetylisoniazid (101).
The ability to
inactivate INH was shown to be inherited in a Mendelian fashion by family pedigree analysis (52) and diminished elimination of INH, an autosomal recessive trait, was found to be the result of reduced enzyme activity (53).
In 1959 it was established that there is a genetic polymorphism in the Arylamine Nacetyltransferase gene in humans for the metabolism of INH (52), and it was in this year as well that the term “pharmacogenetics”, defined as the “study of the role of genetics in drug response” (97) was first formulated by Friedrich Vogel.
In 1960, American
scientists phenotypically classified people as either fast or slow acetylators (fast acetylators being characterised by a homozygous or heterozygous fast allele and slow acetylators as having only the homozygous slow allele) since they found a bimodal
36 distribution in the elimination of INH (52). However, in 1961, Japanese scientists found that the elimination of INH in Japanese populations was distributed in a trimodal fashion, classifying people as fast, intermediate and slow acetylators (150). In 1990, it was shown that there is a definitive correlation in the elimination of INH between the trimodally distributed phenotype and genotype (36).
Thus, humans can be classified as slow,
intermediate and fast metabolisers of INH based on their NAT2 allele type (118).
MOLECULAR MECHANISM OF THE HUMAN N-ACETYLTRANSFERASE ENZYME
Enzymes involved in xenobiotic metabolism are categorised according to their roles: either by changing functional moieties (Phase I enzymes), or by conjugating with an endogenous compound (Phase II enzymes) (152). Isoniazid is principally metabolised by acetylation of a hydrazino or amino group. This reaction is catalysed by Arylamine Nacetyltransferase 2 (NAT2), a cytosolic polymorphic Phase II conjugating enzyme (37,68,119), capable of N-acetylation (usually deactivation), O-acetylation (usually activation) and N,O-acetylation (23). Upon acetylation of INH to acetylisoniazid, by Nacetyltransferase, INH becomes therapeutically inactive. NATs use acetyl Coenzyme A (Ac-CoA) as a cofactor in conjugating an acetyl group onto the amine, hydroxylamine or hydrazine moiety of an aromatic compound (153,171). The metabolic pathway of NAT2 is shown in Figure 2 (115). The acetylated metabolite is usually pharmacologically inactive and less lipid soluble than its precursor, and is excreted (113).
37
Figure 2. Metabolic pathway of NAT2 (115).
38 ACTIVATION OF ISONIAZID BY N-ACETYLTRANSFERASE The activation of the pro-drug INH is altered when the hydrazine moiety has undergone acetylation of INH to acetylisoniazid (ac.INH), and this is responsible for the reduction of therapeutic concentrations of INH (52,154). Recently it was suggested that impaired INH metabolism might be associated with polymorphisms within the NAT2 gene, in a Japanese population (86). It is therefore possible that a fast acetylator genotype (118) can indirectly contribute to the INH resistant phenotype in Mycobacterium tuberculosis by lowering the serum concentration of the drug, since the fast acetylator may not respond to INH in the management of TB (51).
N-ACETYLTRANFERASE GENES
In humans there are three N-acetyltransferase (NAT) loci: two functional genes (NAT1 and NAT2) as well as a pseudogene (NATP) which contains multiple premature stop codons (23,69). The human NAT2 gene encodes for a multi-allelic locus, and the enzyme exhibits functional polymorphism (81,160). Polymorphism in NAT2 activity is one of the most common in the human population, with wide phenotypic and allelic variation within human populations (12,28,60,69).
39 HUMAN ACETYLATION POLYMORPHISMS
Acetylation polymorphisms in humans are due to allelic variation at the NAT2 gene locus and to date (April 2005) there are 36 NAT2 alleles that have been reported (9,69,78,81,160).
Every single allele differs from the fast acetylator NAT2 allele,
NAT2*4, at a combination of between 1 and 4 point mutations all located within the 870 bp coding region. A total of 13 different point mutations distinguish the variant NAT2 alleles from NAT2*4.
Seven missense substitutions (G191A, T341C, A434C, G590A, A803G, A845C, and G857A) and four silent substitutions (T111C, C282T, C481T, and C759T) have been identified in the NAT2 coding region (76). The reference allele NAT2*4 (associated with fast acetylator phenotype) (60,76) is regarded as the wild-type allele since all of the above mentioned substitutions are absent in this allele, although it is not the most common allele in various ethnic groups.
A number of these point mutations are common to more than one NAT2 allele, and are named according to the most common functionally significant nucleotide substitution. Thus, for example, NAT2 alleles containing the T341C mutation are assigned to the NAT2*5 cluster; whereas alleles containing the G590A mutation are assigned to the NAT2*6 cluster. The most frequent polymorphisms, however, are summarised in Table 4.
40 NAT2 Allele Cluster
Nucleotide change
Amino Acid change
NAT2*5
C341T
Ile114Thr
NAT2*6
G590A
Arg197Gln
NAT2*7
G857A
Gly286Glu
NAT2*12
A803G
Lys268Arg
NAT2*14
G191A
Arg64Gln
Table 4. Most common nucleotide and amino acid substitutions found in the NAT2 allele clusters.
The NAT2 gene is multi-allelic and the frequency of the alleles depends on the ethnicity of the population. Ethnicity has proved to play a huge factor in allelic NAT2 frequency distribution and accounts for the high number of fast acetylators observed in Eastern populations (153). PCR-RFLP genotyping methods have been employed in numerous population studies from different cultures (153). Associations between NAT2 genotype and diseases have also been investigated in epidemiological case-control studies (113).
Most of the allelic variance is due to point mutations, e.g. NAT2*5B exhibits three SNPs at nucleotide positions: 341, 481, and 803; NAT2*6A has two point mutations at positions 282 and 590; NAT*13 has a single mutation at position 282; and NAT2*7A has two nucleotide changes at positions 282 and 857 (160). The frequency of the NAT2 alleles in the normal Caucasian population has been well characterised.
41 Seventy-two to 75% of the NAT2 allele variants are accounted for by NAT2*5B and NAT2*6A. The most common allelic variant found in Caucasians is NAT2*5B (4046%) which encodes the slow phenotype, in contrast to the Japanese population where the frequency of the variant is estimated to be 0.5% (27,28,95).
A silent mutation at position 282 are shared by NAT2*6A, NAT2*7B, and NAT2*13. The slow acetylator phenotype is associated with NAT2*5A, NAT2*5B, NAT2*6A, NAT2*7A, NAT2*7B, and NAT2*13, as a result of a decrease in the NAT2 protein amount. However, the activity of the expressed protein from the NAT2*5A, NAT2*5B, and NAT2*5C genes is lower than that of NAT2*6A and NAT2*7B, whereas NAT2*13 has normal activity even though this allele is associated with the slow acetylator phenotype (77,97). Slow acetylator status varies among ethnic groups and has been characterised as follows: 10-20% of Japanese and Canadian Eskimo, 40-70% of Caucasians and African-Americans, and more than 80% in Egyptians (101,153).
Some of the NAT2 variants alleles have not been phenotypically defined, but the majority of those which have been defined, encode for the slow acetylator phenotypes. However, the rare NAT2*12A allele (27) and the rare NAT2*13 allele
(42,77) which are
characterised by mutations A803G and C282T, respectively, encode for the fast acetylator phenotype, thus indicating that not all variant alleles encodes for an enzyme with reduced activity.
Furthermore, it is suggested that the ethnic distribution of the acetylation
polymorphism existed before Palaeolithic splitting of human populations from Africa (95).
42
VARIATION IN SLOW ACETYLATION ALLELE FREQUENCIES
It was shown that individuals which exhibited the slow acetylator phenotype had reduced concentrations of systemic NAT2 protein (70). There are two mechanisms which elucidate this reduction in catalytic activity; a reduction in the expressed protein concentration and a reduction in the stability of the protein (60). The NAT2 allozymes which had missense mutations G191A (R64Q), T341C (I114T), A434C (E145P), G590A (R197Q), A845C (K282T), and G857A (G286T), all exhibited reduced activity. In addition, the most prominent missense mutations T341C, A434C, G590A and G191A, A845C, G857A, showed considerable reductions in protein activity and intrinsic stability, respectively (70). The Arg 64 residue is essential for efficient NAT activity (37), and is of great importance in maintaining the juxtaposition of the catalytic triad residues (141).
An alteration in the highly conserved Arg 64 residue results in the reduced intrinsic stability of the G191A (R64Q) enzyme.
In contrast, the allozymes encoding silent
mutations did not have noteworthy changes in protein catalytic activity. Silent mutations can affect gene transcription, leading to the formation of truncated proteins with reduced or no function. The absence of increased mRNA levels in these allozymes might be due to rapid fragmentation of the protein or its distorted translation (141). The NAT2*12 and NAT2*13 alleles, which respectively contains the mutations A803G and C282T, encode for
43 enzymes whose catalytic activity is consistent with that of the fast acetylator phenotype (70).
The frequencies of slow acetylator-type individuals in three distinct South African population groups (South African Black, South African Caucasian and Cape Coloured) are dissimilar. Those reported for the South African blacks and whites are: q2 = 0.36, q = 0.60 and q2 = 0.55, q = 0.74, respectively. For the Cape Coloured population it was q2 = 0.286 and q = 0.535 (43). The frequencies of fast and slow alleles in the Cape Coloured population might be due to strong eastern influence, since it correlates well with the Filipino population of south-east Asia (43).
In a study that used INH as a metabolic probe, a 100% correlation was shown between NAT2 genotype and NAT2 phenotype (144). However, the heterozygous fast NAT2*4 individuals exhibited a distinct intermediate acetylation ability, which was significantly lower than the rate of acetylation of INH observed in the homozygous fast NAT2*4 individuals. Similar findings were made by using caffeine as a metabolic probe, but it was difficult to differentiate between slow, intermediate and fast acetylators (28). Thus, individuals with a single NAT2*4 allele are generally considered fast NAT2 acetylators (28).
The NAT2 genotype accurately predicts NAT2 acetylator phenotype (82), which indicates that in large-scale investigations where phenotyping assays may be impractical, or may be distorted by disease status and its treatment, genotyping provides reliable
44 prediction of phenotype (113). The knowledge of the acetylation status of patients within a community receiving anti-TB treatment may be crucial to improve the management of TB, given that the acetylator status only becomes relevant where once weekly chemotherapy is administered (49).
MYCOBACTERIAL N-ACETYLTRANSFERASE AND INH RESISTANCE Recently, a NAT homologue was identified in Mycobacterium tuberculosis (31,119,140) and it was found that bacterial nat substrate specificity is similar to that of human NAT2 (135,154). Over-expression of the drug-inactivating NAT enzyme can confer resistance to INH, but nat is not the target of INH (119). In addition, INH may be inactivated by the N-acetyltransferase (NAT) enzyme, but no INH resistance-associated mutations have been described in the bacterial NAT gene (128) and thus the role of the bacterial nat as a major cause of INH resistance in Mycobacterium tuberculosis was difficult to substantiate (154).
SOCIO-ECONOMIC FACTORS WHICH MAY PLAY A ROLE IN ISONIAZID RESISTANCE
TB has the most devastating impact on the poorest countries in the world (130,174). South Africa (SA) is considered by the World Bank as a third world country and in 2002 only six other countries were estimated by the WHO to have more cases of TB than SA
45 (173). The projected incidence is 556 cases per 100 000 population (173). The TB epidemic is further promoted by the rise in the increase of infection with the human immunodeficiency virus (HIV). Infection with HIV is an absolute specific risk factor for TB with both diseases primarily affecting young people in their productive ages (93).
HIV infection in an individual who is already infected with TB poses an increased risk of the development of active TB (from a 10% risk in their lifetime to 7-8% risk per year) (93). Co-infection with HIV leads to the further spread of the TB epidemic and recently it was shown that in SA, 55% of patients with smear and/or culture positive TB were HIV-positive as well (178).
TB is a social disease and has historically always occurred among impoverished communities such as the homeless, undernourished and overcrowded (7), which is characteristic of third world populations.
In a press release from the WHO in 2004 (179), it was stated that there is not only an increase in the prevalence of any mono-resistance in the world, but also an annual MDRTB increase of more than 3000 new cases in SA alone. The increase in prevalence of resistance to anti-TB drugs might be a reflection of the socio-economic environment that communities are subjected to. These conditions in turn, might be favourable for noncompliance which would assist the bacilli to acquire additional resistance which contributes to future MDR-TB (179). Patients commonly stop taking drugs when their symptoms have disappeared, even though the full course of the treatment programme still
46 needs to be completed. MDR-TB usually results from non-compliant treatment (163), and is associated with death rates of up to 80% (129). However, molecular epidemiology shows that transmission is the major contributor to MDR-TB.
Drug resistance is divided into two types: primary and acquired resistance. Primary resistance is defined as resistance in persons who have never received anti-tuberculosis drugs for more than one month, and these patients are initially infected with drugresistant strains (4). Acquired resistance is defined as resistance to anti-tuberculosis drugs, which arises during treatment due to poor compliance or improper management (4). In low socio-economic communities large numbers of people occupy a single room or few rooms in the house thus exposing any children who may be present to pathogens borne by adults. Thus infection from a drug resistant adult source may cause primary resistance in the children. Adults may be infected with a primary drug-resistant strain or acquire resistance during their treatment (56,147).
TB patients are reluctant to inform health care workers about their TB status, due to fear of being ostracised within their community as well as a feeling of guilt for not adhering to the prescribed regimen. Hence, the terms primary and secondary (acquired) resistance are used incorrectly since the patients may have acquired resistance during previous treatment or their strain was resistant to anti-tubercular drugs from the start and not because resistance was acquired due to previous treatment. To clarify the issue, the WHO and International Union Against Tuberculosis and Lung Disease (IUATLD) implemented the terms drug resistance among new cases and drug resistance among
47 previously treated cases (178). Drug resistance among new cases (previously: primary drug resistance) is the presence of drug-resistant strains of M. tuberculosis in a newly diagnosed patient who has never received anti-tuberculosis drugs or has received them for less than one month (178).
Drug resistance among previously treated cases
(previously: acquired drug resistance) is that found in a patient who has previously received at least one month of anti-tuberculosis drug therapy (178).
48 Risk factors for the development of resistance among previously treated cases are thus summarised follows: (2,3,56,85)
1. previous treatment for TB, 2. non-compliance to treatment, 3. inappropriate prescribing and failure to complete the prescribed regimen, 4. poor management of the DOTS programme, and 5. malabsorption of anti-TB drugs
In addition, risk factors for development of resistance among new cases are: (3,26)
1. contact with an individual already infected with drug-resistant TB 2. HIV infection, and 3. immunocompromising conditions other than HIV infection, and 4. malabsorption of anti-TB drugs, due to circumstances e.g. poverty.
Risk factors for the development of INH resistance in the Cape Coloured population in the Western Cape region of South Africa have not yet been established. It was shown that more than 60% of drug-resistant TB resulted from ongoing transmission of an already drug-resistant strain within a suburb in Cape Town (149,162). Results from an ongoing molecular epidemiology study in these communities also showed that isolates in
49 Family 29, which represent the Beijing genotype, are the second most prominent M. tuberculosis family of strains in the local communities (162). Furthermore, the patients infected with Beijing/W resistant isolates are mostly infected with a W-like isolate which is resistant to INH only.
HYPOTHESIS
The high prevalence of INH mono-resistance in Beijing strains of M. tuberculosis in South Africa is due to the interaction of bacterial and/or host related factors.
The purpose of this study is to provide novel insights into the mechanisms whereby the Beijing strain develops INH resistance. There are many risk factors involved in the development of drug resistance in M. tuberculosis, which are pathogen related (such as gene mutations) and/or host related (such as anti-tubercular drug metabolism, clinical adherence to anti-tubercular treatment, immuno-compromising conditions e.g. HIV, socio-economic factors).
OVERALL AIM
To identify risk factors associated with INH resistance which may be pathogen and/or host specific and which may lead to acquisition of MDR-TB with Beijing strains of M. tuberculosis.
50 The focus is on DNA repair (pathogen related), N-acetyltransferase status (host related) and socio-economic status (host related), which may lead to acquisition of INH resistance in Beijing strains of M. tuberculosis.
The studies are described in the following chapters, as manuscripts to be submitted for publication, and are written according to the guidelines of the Journal of Clinical Microbiology as set out in the instructions to authors, unless otherwise stated. Details of the methods used in these chapters will be elaborated on in Chapter Six. The project, including all ethical aspects, was approved by the Ethics Committee (project number 2002/C118).
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51
CHAPTER 2
Mycobacterial DNA Repair and Isoniazid Resistance
52 ABSTRACT
Introduction:
Bacterial strains with defective DNA repair genes exhibit increased
spontaneous mutation rates. Such hypermutators exhibit improved adaptability during demanding times and this allows for the emergence of variant strains, which include drug-resistant strains. Objective: To identify missense mutations in the DNA repair genes mutT2, Rv3908 and ogt in M. tuberculosis strain families. Design: A PCR-based Dot-blot hybridization strategy was used to identify previously reported missense mutations in the mutT2, Rv3908 and ogt genes of M. tuberculosis clinical isolates from different strain families (n = 402). Results: Of the 180 Beijing isolates (drug resistant and -susceptible) 178/180 (98.89 %) possessed the mutant mutT2 genotype, 100% possessed the mutant Rv3908 sequences and 98.89 % (178/180) displayed a mutant ogt genotype. The majority of the other predominating representative M. tuberculosis families (n = 222) had wild-type DNA repair genes. Discussion: The Beijing family, in contrast to the other strain families, primarily had mutant base excision repair genes. Evolutionary relationship analysis of the mutations in these genes suggests that the mutant Beijing isolates shared a common ancestor. However, further evidence is required to substantiate the suggested findings by means of carrying out high throughput sequencing of candidate genes implicated in INH resistance to establish whether there is an increased mutation rate, as well as a Luria-Delbruck fluctuation-test to phenotypically verify the possible increase in mutations. These
53 investigations may elucidate if the organism has a selective advantage by exhibiting faster mutability, compared to that of other M. tuberculosis strains.
54
INTRODUCTION
With the advent of the antibiotic revolution some 50 years ago, the selective pressure on the Mycobacterium has become surprising (2,3) and between 1994 and 1997, multidrugresistant tuberculosis (MDR-TB) became recognised worldwide (3,4).
In M.
tuberculosis, MDR resistance to antibiotics is often a consequence of genomic mutations in genes encoding for catalase-peroxidase (katG) and the β–subunit of ribonucleic acid (RNA) (rpoB) which contribute to INH and RIF resistance, respectively (5,6). Furthermore, the katG gene encodes for a dual-function catalase-peroxidase enzyme (7) which protects the mycobacterial DNA from hydrogen peroxide damage and this further contributes to the survival of the bacilli in the macrophages (7). However, it was found that in one isolate, a deletion at the 3’ end of the katG gene resulted in loss of enzyme activity and the tubercle bacilli thus attained resistance to INH (8).
The most extensive MDR-TB outbreak of Beijing isolates (9,10) occurred among 267 patients in New York, all of whom were co-infected with HIV.
Members of this
genotype may have an increased probability of acquiring drug resistance due to their ability to mutate more rapidly than other strains (11).
It is suggested that the Beijing
strain might have a selective advantage. An increased mutation rate might further allow for the accumulation of drug-resistant mutations, since Streicher et al. reported that an alarming 48% of all drug resistant isolates from this region, mainly associated with the Beijing/W strain, are INH mono-resistant and might be a source for future MDR-TB (10).
55 The survival and replication of the cell is crucial and thus damaged DNA must be repaired. If DNA damage is not repaired it results in mutational changes (12). One possible mechanism of M. tuberculosis to overcome adverse environmental conditions is by means of genetic mutation that generates individual strains with enhanced fitness (13). The frequency of mutation is highly dependent on the environment and this selection process often leads to the acquisition of mutator phenotypes (14).
Mutator phenotypes resulting from antibiotic exposure has been demonstrated in Escherichia coli (E.coli) and Pseudomonas strains that showed an increased mutation rate (15,16). In addition, it has been shown that higher mutation rates, including drugresistance associated mutations, occur in micro-organisms that exhibit mutations in genes responsible for DNA repair (17).
The same holds true for genome instability and
improved adaptability during times of stress (17,18).
In E.coli, powerful mutator genes are usually mutant DNA repair genes that cause point mutations in the genome sequence (18). DNA damage in bacteria induces the increased expression of a number of genes, resulting in a higher survival rate (19). In particular, the major replicative DNA polymerase (DnaE2) in M. tuberculosis is upregulated by DNA damaging agents (13), which further enhances the mutation frequency and survival of M. tuberculosis. DnaE2 induces mutations that confer resistance to anti-tuberculosis agents. In vivo studies on mice infected with M. tuberculosis, and treated with DNAdamaging agents found similar results from isolates (13).
56 Errors in DNA replication are major sources of spontaneous mutations (20). The genome of M. tuberculosis possesses hypothetical open reading frames (ORF), which are similar to genes known to be involved in repair of DNA lesions due to oxidation and alkylation of nucleotides (21). A BLAST search between putative genes within the E.coli and the mycobacterial genome sequence found that two genes in M. tuberculosis, mutT2 and Rv3908, were homologous to that of the E.coli mutT gene, and a second E.coli gene, ogt, had an ORF in M. tuberculosis (21). These two genes encode for DNA repair enzymes in E.coli. The mutT gene product hydrolyses 8-oxo-deoxyguanisine triphosphate (8-oxoG), a potent mutagenic substrate, in the nucleotide pool to a monophosphate and the ogt gene product removes methyl groups from O6-methylguanine in DNA (21).
The enzymatic function of the mutT protein prevents replicational errors, i.e. the incorporation of the 8-oxoG oxidation product into the newly synthesized DNA. However, in the absence of the functional mutT protein, the oxidative 8-oxoG can basepair with adenine (dA) instead of cytosine (dC) residues of the template strand, thereby promoting AT to GC transversions. This means that by eliminating the oxidized form of guanine nucleotides from the nucleotide pool, that high fidelity of newly synthesized DNA is maintained (18,20). Consequently, disruption of these genes may cause mutator phenotypes for mutant DNA repair genes involved in 8-oxoG excision (22) and is part of the Base Excision Repair system which is of high importance in intracellular pathogens as they are exposed to host-produced Reactive Oxygen Intermediates (ROI) that modify DNA (18).
57 The ability of the widespread MDR Beijing strains to adapt to adverse conditions and thereby enhancing their virulence is indicative of an accumulation of genomic mutations associated with resistance (21). Furthermore, the inability of any bacterial strain to successfully carry out DNA repair, subsequently forces the bacterium to increase its mutation rate (14). Thus the bacterium would benefit from an increased mutation rate, resulting in a hypermutative state of the organism, accompanied by an increase in single nucleotide polymorphisms (SNPs). However, it is suggested that most of the nucleotide changes might be silent, since undesirable mutations would be deleterious.
M. tuberculosis has about 1 synonymous difference per 10 000 synonymous sites (23) and there is a remarkable reduction in silent nucleotide substitutions in M. tuberculosis (24) compared to that of other human pathogenic bacteria. The estimation of 1 SNP per 10 000 synonymous sites was only obtained from SNPs that resulted in silent substitutions. SNPs that were located in putative regulatory regions were not included since it is believed that these SNP are the result of selective pressure (25). It is hypothesised that DNA damage repair gene polymorphisms may improve mycobacterial adaptation to hostile environments.
At the time when this study was initiated, a paper appeared in the literature which stated that the acquisition of alterations in genes involved in the repair of DNA mutations (mut genes) may explain why the Beijing strains have an ability to adapt to their environment (21). The authors found that the Beijing/W genotype strains exhibited unique missense mutations in three putative mut genes and these in turn were characteristic of this particular strain family. The aim of the current study is to identify genotypic differences
58 in M. tuberculosis strain families by means of screening for mutations in the DNA repair genes mutT2, Rv3908 and ogt which may further the acquisition of INH resistance.
59
MATERIALS AND METHODS
CLINICAL ISOLATES
The clinical isolates used in this study were from well characterised isolates from the Medical Biochemistry Database. Different M. tuberculosis strain families were chosen to screen for mutations which may contribute to drug resistance including the Beijing strain which predominates within the study region. Isolates were recovered from patients with culture-tested drug-resistant and drug-susceptible tuberculosis. The Beijing Family isolates, Family 29, predominating from the 72 designated clinics, constitutes one of the four prominent M. tuberculosis strain families. Family 28, Family 11 and the Low Copynumber Clade (L.C.C), constitutes the other three strain families investigated.
The
collection of M. tuberculosis isolates (n = 402) consisted of 180 (44.8%) Beijing strains (isolates resistant to one or more drugs included: n = 87 for isolates from the 72 clinics in the Boland-Overberg and Southern Cape-Karoo (BOKS) regions of the Western Cape Province in South Africa ; n = 13 for isolates from Gugulethu; n = 11 for isolates from Zimbabwe; n = 10 for isolates from Welkom; n = 8 from isolates from Cape Town; n = 1 for an isolate from Uganda; and drug susceptible isolates consisted of 50 isolates originating from two communities [Ravensmead and Uitsig] in Cape Town). Twelve (3%) atypical (ancestral) Beijing strains, which diverged early in the evolution of the Beijing phylogenetic lineage (21), were included as well. The Atypical Beijing strains have different RFLP patterns to those of the Beijing strains, but share identical spoligotype patterns. Seventy-two isolates from strain family 28 (18%); 77 isolates from
60 strain family 11 (19.1%); and 61 isolates of the LCC strain family (15.1%) were from Ravensmead / Uitsig as well.
Isolates from 72 clinics in the BOKS regions were collected over a two year period and were all subjected to culture and drug susceptibility testing (DST), assessed by the indirect proportion method on Löwenstein-Jensen medium (26). The isolates from the two communities in Cape Town, of which the phenotypes are as yet unknown, were collected between January 1999 and January 2000. Spoligotyping of all the samples were done as previously described (27,28). One-hundred-and-sixty-four of the isolates were previously grouped into strain families based on IS6110 restriction fragment length profiles by standardised methods (29).
According to their RFLP pattern, isolates can be characterised into clusters. Selection criteria of representative clusters within each family included:
(i)
the most prominent clusters, and
(ii)
having two or more clusters if the clusters were highly diverse within a particular family.
The most prominent clusters in the Beijing strain family are clusters 208 (n = 13), 209 (n = 6), 210 (n = 5) and 220 (n = 29). Seventeen (58%) of the Beijing family isolates which belonged to the largest cluster, cluster 220, are INH mono-resistant.
61 Unique clusters consisted of isolates whose RFLP patterns were highly diverse and scattered. Similarly, isolates from Family 28, Family 11 and the L.C.C. Family, were selected according to their clustering characteristics.
The representative number of
isolates per strain family as well as cluster representation is listed in Table 1. The reference strain H37Rv was used in all experiments as a control.
PCR AMPLIFICATION
DNA templates from all the isolates (n = 402) were available. Primers were designed, according to the DNA sequences of the mutT2, Rv3908 and ogt genes which were obtained from the sequence database at the TubercuList website (30), to amplify the three putative mutator genes in M. tuberculosis. The primers are given in Table 2.
DNA amplification of the three genes separately, was executed in a 100μl reaction by making use of 100ng of genomic DNA as template, 10 μl of a 10X Taq DNA polymerase buffer (Qiagen), 20 µl of a 1X Q solution buffer (Qiagen), 25 mM MgCl2, 0.8 mM dNTP’s (dATP, dCTP, dGTP and dTTP), 0.3 µM (10pmol/L stock) of the separate sets of oligonucleotide primers (Table 2.2), 2.5 U (0.5 μl) Hotstart Taq DNA polymerase (5 U/µL) and 50 µl water.
PCR amplification was executed via thermal cycling in an Eppendorf Mastercycler epgradient S (Merck). The cycling parameters and PCR conditions are shown in Table 3.
62 A blank control, containing all the reagents with the exception of DNA, was included in each reaction to ensure that no cross contamination occurred whilst carrying out the PCR procedure in order to ensure quality control of the amplified samples. The expected amplification products of the three genes were visualised on a 1.5% agarose gel in 1xTBE buffer with ethidium bromide using a previously described method (31).
PCR DOT-BLOT HYBRIDISATION
Allele-specific wild-type and mutant oligonucleotide probes for all three genes involved in DNA repair were designed, according to the position of the missense mutations in the three genes as described by Rad et al (21). The probes are given in Table 4. A PCRbased dot-blot hybridization strategy (31) was used to screen for specific missense mutations in the mutT2, Rv3908 and ogt genes of all clinical isolates from various strain families. Amplified DNA of all three genes from the reference strain H37Rv, was included on each blot to indicate the wild-type control as well as amplified DNA from a well characterised isolate known to have the mutant gene. All three individually fixed dot-blot membranes, containing the loaded amplification products of the separate genes for all isolates, were initially hybridised with the mutant-specific radiolabelled probes which was followed by subsequent hybridisation with the wild-type-specific probes after stripping of the blots.
Autoradiography was done after hybridization to carry out
radioisotopic detection.
The radiolabelled membranes were initially overexposed to
ensure efficient blotting of the amplified products.
63 Briefly, oligonucleotide probes were radioactively labelled at the 5’ end by phosphorylation with [γ-32P]-ATP (Amersham) as described previously (31). A 15µl aliquot of each PCR product was mixed with 140 µl dot-blot buffer (0.4M sodium hydroxide, 25 mM EDTA) and denatured at 95˚C for 5 min. Afterwards the denatured PCR product was immediately loaded, under vacuum, onto a Hybond-N+ nylon nitrocellulose membrane (Amersham) in a dot-blot apparatus (Biorad) to which 150 µl dot-blot buffer had been previously added. The DNA was fixed onto the membrane by baking it at 80˚C for 2 hours.
Pre-hybridisation of the nitrocellulose membrane was carried out at 10˚C below the Tm of each probe for 30 min using 7.2 ml 5xSSPE, 0.4 ml Denhardts and 0.4 ml 10% SDS. Fifty micro-litres of the probes were added to the pre-hybridisation buffer. Hybridization was carried out for 1 hour. For radioisotopic detection, each filter was hybridized in 5xSSPE and finally washed in 1.5xSSPE for 10 min at 74˚C as previously described (31). Autoradiography was done with X-ray film for 16 hours at -80˚C. After blotting with the Wild-type probes, the membranes were stripped in 0.4M NaOH for 45 min by incubating the membranes at 50˚C and neutralised for 30 min at 42˚C to carry out subsequent probing with the mutant specific probes. Overexposure of all first time autoradiographs were done to ensure sufficient blotting of all samples and afterwards a final autoradiograph was done.
64 SEQUENCE ANALYSIS
Direct sequencing of representative PCR products with the corresponding primers (1.1µM) was done with an ABI PRISM® 3100-AVANT Genetic Analyzer (Perkin Elmer) to verify the results obtained from dot-blot analysis.
GENOME SEQUENCE DATA AND ANALYSIS
The genomes of three strains: M. tuberculosis H37Rv, M. tuberculosis CDC 1551, and M. tuberculosis 210, belonging to principal genetic group (PGG) 1, 2, and 3, respectively, were compared by means of in silico analysis. A Bioinformatics search on the three putative mutator genes: mutT2, Rv3908, and ogt, as well as four candidate genes which are possibly involved in INH resistance (iniB, efpA, accD6 and Rv1592c) was done to determine the frequency of Single Nucleotide Polymorphisms within these genes in M. tuberculosis. These four genes were selected to only serve as an indicator of the amount of SNPs between the three genomes of M. tuberculosis. The sequences of the individual genes were obtained from Tuberculist (30). The complete genome sequences of H37Rv and CDC 1551 was obtained from the publicly available finished genome sequence databases from the Sanger Centre / Pasteur Institute and The Institute for Genomic Research (TIGR), respectively (30,32-35) and the preliminary sequence data for M. tuberculosis 210 was obtained from the unfinished genome sequence database from the TIGR website (36). BLAST similarity searches (37) were done to screen for SNPs within these genes between the various genome sequences.
65
STATISTICAL ANALYSIS
Statistical analysis was performed using STATISTICA version 7 (38). Two by two contingency tables were drawn up by means of cross-tabulation and the observed frequencies of all three genes were calculated by means of the Pearson’s Chi-squared test.
DENDOGRAM FORMATION
Of the 87 Beijing isolates from the 72 clinics in the Western Cape Province, a total of 80 isolates was previously characterised by internationally standardised IS6110 RFLP fingerprinting protocols (39) and subsequently classified into clusters. These fingerprints were compared using GelCompar II software (Applied Maths, Kortrijk, Belgium) to generate a dendogram that reflected the evolutionary relationships of the Beijing isolates investigated. A construction of the dendogram is shown in Figure 1.
66
RESULTS
PCR AMPLIFICATION OF THE mutT2, Rv3908 AND ogt GENES INVOLVED IN DNA REPAIR
Successful PCR amplification of the three genes involved in DNA repair was confirmed on 1.5% agarose gels. Comparison of the resulting amplification products with the IX Marker (0.072 – 1.35 kbp) revealed that the expected bands were the correct size fragments. Examples of mutT2, Rv3908 and ogt amplification products are displayed in Figure 2.
DOT-BLOT ANALYSIS
Representative examples of mutT2, Rv3908 and ogt mutant-specific dot-blots, as well as the wild-type-specific dot-blot of mutT2 for F11, are displayed in Figure 3. Results of the other wild-type-specific dot-blots are not shown.
MUTATIONS IN DNA REPAIR GENES OF M. TUBERCULOSIS
Allele-specific probing revealed that 178/180 (98.89 %) of all the Beijing strains (drug resistant and drug susceptible) contained mutant mutT2 genes, 100 % contained a mutant Rv3908 gene and 178/180 (98.89 %) contained a mutant ogt gene. The mutational data off all the samples investigated are given in Appendix A. It was expected that all of the
67 Beijing isolates would give a positive hybridisation signal with the mutant-specific probes for all three genes.
However, 2/180 (1.1 %) of the isolates exhibited the wild-type gene for both mutT2 and ogt. Both wild-type genes were present in the same isolate which originated from Cape Town (S1116). Isolate R321 only exhibited the wild-type mutT2 gene and isolate R78 exhibited the wild-type ogt gene. Similarly, an isolate from strain family 28 which was expected to have the wild-type Rv3908 gene, exhibited the contrary. Automated DNA sequencing confirmed the discrepant results. The IS6110 RFLP patterns, spoligotype patterns and all other associated INH resistance mutations of Beijing isolates within the US Medical Biochemistry computer database showed that there were no clear distinguishable features which may give insight into why discrepant results were obtained.
In contrast, the Atypical Beijing isolates had similar results with regards to the wild-type genes for mutT2 and ogt, except for Rv3908 where only 9/12 (75%) isolates had the wild-type gene. Other prominent strain families (F28, F11 and the L.C.C.) had wild-type genes. Seventy-one out of 72 isolates (96.8%) of Family 28 had wild-type genes for both mutT2 and Rv3908 and all isolates (100%) had the wild-type ogt gene. All isolates from Family 11 and the L.C.C. had the wild-type genes.
68 One isolate however, exhibited a positive hybridisation signal with both the mutant and wild-type probes for mutT2 and Rv3908, and a positive signal for the ogt wild-type probe. This finding might represent a case of dual infection.
The cross-tabulation results for the three genes, indicated that there was a significant difference (p51 unknown Black Coloured White Asian Female Male Positive Negative Unknown Yes No Yes No Yes No Abstain Yes Unknown No Yes Unknown R5000 / m Unknown Full-time Maintenance Part-time Pension Unemployed Unknown
212 n 17 42 64 42 25 22 36.58 39 173 0 0 90 122 39 151 22 10 202 5 207 52 160 99 91 22 70 122 20 116 73 23 15 66 77 32 0 0 22 102 65 16 6 0 1 0 22 43 31 11 5 100 22
% 8.9 22.1 33.7 22.1 13.2 18.4 81.6 0 0 42.4 57.6 20.5 79.5 4.7 95.3 2.4 97.6 24.5 75.5 17.4 82.6 36.4 63.6 61.4 38.6 7.9 34.7 40.5 16.8 0 0 53.7 34.2 8.4 3.1 0 0.5 0 22.6 16.3 5.8 2.6 52.6 -
INH-mono Resistant 52 n % 0 0 14 32.5 15 34.9 10 23.3 4 9.3 9 35.32 8 15.3 44 84.7 0 0 0 0 18 34.6 34 65.4 4 7.7 48 92.3 0 0 2 3.8 50 96.2 1 1.9 51 98.1 14 27.0 38 73.0 30 57.7 22 42.3 0 0 23 44.2 29 55.8 0 0 29 66.0 15 34 8 4 9.0 14 31.1 25 55.5 2 4.4 0 0 0 0 7 22 48.9 20 44.4 2 4.4 1 2.2 0 0 0 0 0 0 7 9 20.0 8 17.8 7 15.5 2 4.4 19 42.2 7 -
MDR 52 n % 1 2.7 9 24.3 17 45.9 8 21.6 2 5.4 15 34.55 7 13.4 45 86.5 0 0 0 0 17 32.7 35 67.3 2 3.8 50 96.2 0 0 7 13.4 45 86.6 1 1.9 51 98.1 13 25.0 39 75.0 35 67.3 17 32.7 0 0 23 44.2 29 55.8 0 0 24 68.5 11 31.5 17 5 13.5 16 43.2 16 43.2 0 0 0 0 0 0 15 15 40.5 20 54.0 2 5.4 0 0 0 0 0 0 0 0 15 10 27.0 2 5.4 11 29.7 0 0 14 37.8 15 -
Other 15 n % 0 0 1 10.0 6 60.0 2 20.0 1 10.0 5 35.62 0 0 14 93.3 1 6.7 0 0 6 40.0 9 60.0 3 20.0 12 80.0 0 0 1 6.7 14 93.3 1 6.7 14 93.3 1 6.7 14 93.3 11 73.3 4 26.7 0 0 8 53.3 7 46.7 0 0 5 71.4 2 28.6 8 2 28.6 2 28.6 2 28.6 1 14.3 0 0 0 0 8 2 28.6 5 71.4 0 0 0 0 0 0 0 0 0 0 8 2 28.6 3 42.8 0 0 0 0 2 28.6 8 -
†: Kruskal-Wallis test; §: Pearson’s Chi-squared test ‡ Overall Fisher's Exact test *: p-value indicates a significant difference (p 5 years
7
Don't Know
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9
Not Applicable
Q29. Have you had TB before?
Q30. How many times before? (Choose one)
1
Yes
0
No
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Not Applicable
0
1 times
1
2 times
2
3 times
3
4 times
4
>4 times
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8
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Not Applicable
1
Yes
0
No
7
Don't Know
8
Refuse to Answer
9
Not Applicable
Q31. Did you complete your treatment at all times?
