Susceptibility testing and resistance mechanisms to second-line agents against Mycobacterium ...

191 Pages·2017·8.01 MB·English

Susceptibility testing and resistance mechanisms to second-line agents against
Mycobacterium tuberculosis in Pretoria, South Africa

by

HALIMA M SAID

Submitted in the partial fulfilment of the requirements for the degree

PHILOSOPHIAE DOCTOR
PhD (MEDICAL MICROBIOLOGY)

In the

Department of Medical Microbiology
Faculty of Health Sciences
University of Pretoria

May 2012

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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 by me in respect of a
degree at any other University or tertiary institution.

Signed: ………………this ……………..day of ………………2012

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“A dream doesn't become reality through magic; it takes sweat, determination and hard
work”
Colin Powell

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ACKNOWLEDGEMENTS

First and foremost I would like to thank God (Allah) for blessing me and helping me finish
my degree. Without Allah’s help and blessing the completion of this study would not have
been possible.
I wish to express my sincere gratitude to my supervisor, Prof Ehlers for her supervision,
advice, and suggestions in formulating and planning of the research project as well as reading
and correcting my manuscript to the completion. Working with her was a privilege. Without
her help this study would not have been possible.
I also wish to express my gratitude to my co-supervisor, Dr Kock for her very valuable
support, suggestions and sharing her skills with me in analyzing my sequencing results as
well as her active role in all aspects of the project including correcting of my manuscript.
I would like also to thank Prof Hoosen for his valuable input, advice and guidance from the
very early stage of this research to the end. I always will be grateful for the opportunities he
have opened for me and giving me extraordinary experiences through out my studies. Without
his daily encouragement and support this study would not have been possible.
My appreciation extends no less to Dr Ismail for his valuable input, suggestions and his
crucial contribution to the project from the early stage of the project to the end.

Last but not least I would like to express my appreciation to the following people who made

the research project possible:

(cid:1) Dr Baba for his valuable input and suggestions
(cid:1) Mrs Matsie Mphahlele for sharing her skills on spoligotyping with me and supervising
my laboratory work. I am grateful in every possible way and hope to keep up our
collaboration in the future
(cid:1) The staff members of NHLS laboratory in Medunsa for their assistance in specimen
and data collection
(cid:1) The staff members of NHLS laboratory at the Tshwane Academic Division for their
assistance and allowing me to use the TB laboratory for culture and drug susceptibility
testing
(cid:1) Dr Peter De Mayer for his assistance in analyzing the spoligotyping results
(cid:1) Prof Fanus Venter for allowing me to use his facility for my data analysis
(cid:1) My family and friends for their love and support

Finally, I would like to thank everybody who was important to the successful realisation of
this thesis, as well as expressing my apology that I could not mention personally one by one.
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TABLE OF CONTENT

Page

SUMMARY v

LIST OF ABBREVIATIONS vii

LIST OF TABLES ix

LIST OF FIGURES x

LIST OF PUBLICATIONS AND CONFERENCE CONTRIBUTIONS xi

CHAPTER 1: INTRODUCTION 1

CHAPTER 2: LITERATURE REVIEW 7

2.1 Introduction 7
2.2 Classification of mycobacteria 8
2.3 General characteristics of mycobacteria 9
2.4 Virulence factors of M. tuberculosis 10
2.5 Pathogenesis of M. tuberculosis 10
2.6 Treatment of TB 11
2.7 Drug-resistance in M. tuberculosis 13
2.8 Epidemiology of drug-resistant TB 14
2.9 Genetic basis of resistance against first and second line anti-TB drugs 14
2.9.1 Isoniazid 16
2.9.2 Rifampicin 18

2.9.3 Pyrazinamide 19
2.9.4 Streptomycin 20
2.9.5 Ethambutol 20
2.9.6 Kanamycin, amikacin, capreomycin and viomycin 22
2.9.7 Fluoroquinolones 22
2.9.8 Ethionamide 24
2.9.9 Serine analogues 24
2.10 Diagnosis of drug-resistant TB 25
2.10.1 Phenotypic susceptibility testing techniques for M. tuberculosis 25
2.10.1.1 Conventional solid culture-based susceptibility testing methods for M. tuberculosis 25
2.10.1.1 Rapid solid culture-based susceptibility testing methods for M. tuberculosis 26
2.10.1.2 Liquid culture-based susceptibility testing methods for M. tuberculosis 28
2.10.2 Molecular detection of drug-resistance in M. tuberculosis 31
2.10.3 Molecular epidemiology of M. tuberculosis 35
2.10.4 Genotyping methods used for M. tuberculosis 35
2.10.4.1 IS6110-RFLP genotyping of M. tuberculosis 35
2.10.4.2 Spoligotyping of M. tuberculosis 36
2.10.4.3 Mycobacterial interspersed repetitive unit-variable number tandem 37
repeat typing of M. tuberculosis
2.10.5 Strain families of M. tuberculosis 39
2.10.5.1 The East African-Indian family 40
2.10.5.2 The Beijing family 41
2.10.5.3 The CAS or Delhi lineage 43
2.10.5.4 The Haarlem family 43
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2.10.5.5 The LAM family 44
2.10.5.6 The X family 44
2.10.5.7 The T families 44
2.11 Summary 45
References 47

CHAPTER 3: COMPARISON BETWEEN THE BACTEC MGIT 960 SYSTEM AND 75
THE AGAR PROPORTION METHOD FOR SUSCEPTIBILITY
TESTING OF MULTIDRUG RESISTANT TUBERCULOSIS STRAINS
IN A HIGH BURDEN SETTING OF SOUTH AFRICA

3.1 Background 76
3.2 Results 77
3.3 Discussion 78
3.4 Methods 80
3.4.1 Study design 80
3.4.2 Specimens 80
3.4.3 Routine drug susceptibility testing using BACTEC MGIT 960 80
3.4.4 Drug susceptibility testing using the agar proportion method 81
3.4.5 Analysis 81
Acknowledgements 81
Reference 82

CHAPTER4: EVALUATION OF THE GENOTYPE® MTBDRSL ASSAY FOR 86
SUSCEPTIBILITY TESTING OF SECOND-LINE ANTI
TUBERCULOSIS DRUGS

4.1 Introduction 87
4.2 Materials and Methods 88

4.2.1 Study setting and clinical isolates 88
4.2.2 Drug susceptibility testing 88
4.2.3 GenoType® MTBDRsl assay 88
4.2.4 DNA sequencing of discrepant results 88
4.2.5 Statistical analysis 88
4.3 Results 89
4.4 Discussion 91
Acknowledgements 93
References 94

CHAPTER 5: MOLECULAR CHARACTERIZATION AND SECOND-LINE ANTI-TB 98
DRUG-RESISTANCE PATTERNS OF MULTIDRUG RESISTANT
TUBERCULOSIS ISOLATES FROM THE NORTHERN REGION OF
SOUTH AFRICA

4.1 Introduction 99
4.2 Materials and Methods 100
5.2.1 Study population 100
5.2.2 Routine culture and drug susceptibility testing 101
5.2.3 Drug susceptibility testing 101
5.2.4 Genotyping 102
5.2.5 Data analysis 102
5.3 Results 103
5.4 Discussion 105
5.5 Conclusions 107
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Acknowledgements 108
References 108

CHAPTER 6: CONCLUDING REMARKS 126

Future research 132
References 134

APPENDIX A: DETAILED METHODOLOGY 138

APPENDIX B: DATA AND DETAILED RESULTS 147

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SUSCEPTIBILITY TESTING AND RESISTANCE MECHANISMS TO
SECOND-LINE AGENTS AGAINST MYCOBACTERIUM TUBERCULOSIS IN
PRETORIA, SOUTH AFRICA

by

HALIMA MOHAMMED SAID

PROMOTER: Prof MM Ehlers (University of Pretoria/NHLS)
CO- PROMOTER: Dr MM Kock (University of Pretoria/NHLS)
DEPARTMENT: Medical Microbiology, Faculty of Health Sciences, University of
Pretoria
DEGREE: PhD (Medical Microbiology)

SUMMARY

Multidrug-resistant (MDR) and extensively drug-resistant tuberculosis (XDR-TB) is a serious
problem in South Africa. Standardised and accurate drug susceptibility testing for first-line
and second-line anti-TB drugs are critical elements in battling the MDR- and XDR-TB
epidemic. Furthermore, u nderstanding of the population structure and transmission patterns
of circulating drug-resista nt Mycobacterium tuberculosis (M. tuberculosis) strains is of utmost
importance. Previous studies have shown that different genotypes of M. tuberculosis strains
predominate in different geographical regions and strain to strain variations may have
important consequences when it comes to transmissibility, diagnostics and management.
Despite the high prevalence of MDR- and XDR-TB in South Africa, data with regard to the
population structure of drug-resistant strains is lacking in many regions. Therefore, the aim of
the study was to evaluate phenotypic and genotypic methods for susceptibility testing of first-
line and second-line anti-TB drugs and molecular characterisation of drug-resistant strains
from high-burden TB areas in South Africa.

Consecutive MDR-TB isolates from four provinces were included in this study. The results
of routine drug susceptibility testing of ethambutol, streptomycin, kanamycin and ofloxacin
using the BACTEC MGIT 960 system in MDR-TB isolates was compared to the agar
proportion method. The GenoType® MTBDRsl assay was evaluated for the detection of
resistance against ethambutol, capreomycin, kanamycin and ofloxacin. Finally, the study
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investigated the population structure and transmission patterns of drug-resistant
M. tuberculosis isolates using spoligotyping and MIRU-VNTR typing. Additionally, the
possible association of genotypes with drug-resistance patterns and demographic information
were explored.

The sensitivity and specificity of the BACTEC MGIT 960 system using the agar proportion
method as a gold standard was 18.8% and 96.5% for ethambutol, 95.2% and 37.4% for
streptomycin, 54.6% and 91.8% for kanamycin and 100% and 89% for ofloxacin,
respectively.

The GenoType® MTBDRsl assay gave comparable results for ofloxacin; however, a low
performance was found for the detection of resistance to capreomycin, kanamycin and
ethambutol. In order to resolve the discrepancies between the two methods, DNA sequencing
was done for the target genes (gyrA, rrs, embB) from the discrepant results as well as two
additional genes (gyrB, tlyA) that were not included in the assay. The DNA sequencing
identified mutations in the gyrA, gyrB, rrs and tlyA genes that were not detected by the
GenoType® MTBDRsl assay.

The prevalence of XDR-TB and pre-XDR TB was 7.1% and 9.5%, respectively. A high-

diversity of M. tuberculosis strains were found in this study, with the Beijing and EAI1_SOM

families being predominant. No association was found between genotypes and specific drug-
resistance or demographic information.

The sensitivity of the BACTEC MGIT 960 system for ofloxacin and streptomycin was
excellent; however, the sensitivity was low for ethambutol and kanamycin. The GenoType®
MTBDRsl assay was promising for the detection of ofloxacin; however, the sensitivity of the
assay needs to be improved for capreomycin, kanamycin and ethambutol. The high-level of
diversity and the geographical distribution of the drug-resistant M. tuberculosis isolates in this
study suggested that the transmission of drug-resistant TB in these study settings is not caused
by clonal spread of a specific M. tuberculosis strain.

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LIST OF ABBREVIATIONS

AMK Amikacin
AP Alkaline phosphotase
BCG Bacillus Calmette-Guérin
CAP Capreomycin
CIP Ciprofloxacin
CLSI Clinical and Laboratory Standards Institute
DOTS Directly Observed Treatment Short course
DR Direct Repeats
DST Drug Susceptibility Testing
EAI East African Indian
EMB Ethambutol
ERDR EmbB Resistance Determining Region
ETH Ethionamide
FIND Foundation for Innovative New Diagnostics
FLQ Fluoroquinolone
FMO Flavin Monooxygenase
FRET Fluorescent Resonant Energy Transfer
GI Growth Index
GLI Global Laboratory Initiative
H Haarlem
HIV Human Immunodeficiency Virus
HPLC High Performance Liquid Chromatography
INH Isoniazid
IS Insertion Sequence
IUATLD International Union Against Tuberculosis and Lung Disease
KAN Kanamycin
LAM Latin American Mediterranean
LJ Löwenstein-Jensen
LSP Large Sequence Polymorphisms
M. africanum Mycobacterium africanum
M. bovis Mycobacterium bovis
M. tuberculoisis Mycobacterium tuberculosis
MDR-TB Multi Drug-Resistant Tuberculosis
MGIT Mycobacteria Growth Indicator Tube
MIRU-VNTR Mycobacterial Interspersed Repetitive Unit-Variable Number of Tandem
Repeats
MODS Microscopic Observation Drug Susceptibility
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