siphiwe ruthy matukane

90 Pages·2016·1.23 MB·English

Association of Mycobacterium tuberculosis genotypes and treatment outcome in pulmonary
tuberculosis patients in Tshwane Metropolitan area

SIPHIWE RUTHY MATUKANE
Association of Mycobacterium tuberculosis genotypes and treatment outcome in pulmonary
tuberculosis patients in Tshwane Metropolitan area

by

SIPHIWE RUTHY MATUKANE

Submitted on partial fulfilment of the requirement for the degree

MAGISTER SCIENTIAE
MSc (MEDICAL MICROBIOLOGY)
In the
Department of Medical Microbiology
Faculty of Health Sciences
University of Pretoria
Pretoria
South Africa

September 2015
DECLARATION

I, the undersigned, declare that the dissertation hereby submitted to the University of Pretoria for
the degree MSc (Medical Microbiology) and the work contained therein is my own original work
and has not previously, in its entity or in part, been submitted to any University for a degree.

Signed:……….………………..this……………………………day of………………………2015
I can do all things through
Christ,
He strengthens me…
ACKNOWLEDGEMENTS
I would like to sincerely thank:
National Research Foundation (NRF): For providing funding for my MSc degree for which
without them it would not have been possible.
Dr MR Lekalakala, Department of Medical Microbiology, University of Pretoria, for her
professional supervision in the successful completion of this research project, moreover for her
guidance, care, understanding and patience and for always going the extra mile in assisting me.
Dr HM Said, Department of Medical Microbiology, University of Pretoria, for her molecular
biology expertise, technical expertise and co-supervision regarding this project and for all your
professional guidance, support and for all that you continue to teach me.
Dr N Ismail (Head of Department) National Institute of Communicable diseases
Tuberculosis Referral Laboratory, for letting me carry out my entire laboratory work at the
laboratories.
Prof MN Mbelle (Head of Department) Department of Medical Microbiology, University of
Pretoria, for all her support and always ready to listen and try to help.
My colleagues: Nakedi and Ruth (for proofreading my work), for all their support and
friendship which carried me through my MSc studies.
My family: mother (Nester), my two sisters (Busisiwe and Kurhula), my grandmother
(Emma),my cousin (Lucern) and my partner (Selby) for their consistent unconditional love,
support, confidence in me and all the encouragement I always receive from all of you.
And most importantly GOD, when I felt I could not do it. He gave me strength, confidence and
courage to do it, see it through and finish my work, no matter what, no matter how long it took.
TABLE OF CONTENTS
PAGE

LIST OF TABLES i
LIST OF FIGURES ii
LIST OF ABBREVIATIONS iii
LIST OF PUBLICATIONS AND PRESENTATIONS v
SUMMARY vi
CHAPTER 1: INTRODUCTION 1
AIM 4
OBJECTIVES 4
CHAPTER 2: LITERATURE REVIEW 5

2.1. Introduction 5
2.2 Historical background of tuberculosis 6
2.3 Classification and characteristics of Mycobacteria 7
2.4 Pathogenesis of M. tuberculosis 7
2.5 Immunity to tuberculosis 8
2.6 Virulence factors of M. tuberculosis 9
2.7 Epidemiology M. tuberculosis 10
2.7.1 Tuberculosis in South Africa 10
2.8 Development of drug-resistant tuberculosis 11
2.9 Diagnosis and detection of drug resistance of M. tuberculosis 12
2.9.1 Clinical detection 13
2.9.2 Microscopy detection of M. tuberculosis 13
2.9.3 Solid culture based methods 13
2.9.3.1 Lowenstein Jensen culture media 14
2.9.3.2 Middlebrook Media 14
2.9.4 Liquid culture based methods 15
2.9.4.1 The BACTEC 960 MGIT system 15
2.9.4.2 Versa TREK System 16
2.9.5 Molecular based assays for the detection and identification of M. tuberculosis 16
2.9.5.1 Nucleic acid amplification technique (NAAT) 16
2.9.5.2 Line probe assay 17
2.9.5.3 INNO-LiPA® MYCOBACTERIA V2 17
2.9.5.4 The Genotype®MTBDRplus assay 18
2.9.5.5 GeneXpert® system 18
2.9.6 Serological assays for the detection of mycobacteria 18
2.9.6.1 IGRAS-Interferon-Gamma Release Assays 19
2.9.7 Differentiation of mycobacteria 19
2.10 Treatment of tuberculosis 21
2.11 Control and eradication of tuberculosis 22
2.12 Vaccines 22
2.13 Molecular epidemiology of M. tuberculosis 23
2.14 Restriction Fragment Length Polymorphism 23
2.15 Mycobacterial Interspersed Repetitive Units-Variable Number 24
of Tandem Repeats
2.16 Spacer oligonucleotide typing 27
2.17 Treatment outcome associated with different M. tuberculosis genotypes 30

CHAPTER 3 ASSOCIATION OF MYCOBACTERIUM TUBERCULOSIS
GENOTYPES AND TREATMENT OUTCOME IN
PULMONARY TUBERCULOSIS PATIENTS IN
TSHWANE METROPOLITAN AREA 29

3.1 Abstract 31
3.2 Background 33
3.3 Materials and methods 34
3.3.1 Clinical isolates and setting 34
3.3.2 Data collection 35
3.3.3 DNA extraction 35
3.3.4 Spoligotyping 35
3.3.5 MIRU-VNTR typing 36
3.3.6 Quality Control 36
3.3.7 Statistical analysis 37
3.3.8 Ethical approval 37

CHAPTER 4 RESULTS AND DISCUSSION 38

4.1 Results 38
4.1.1 Spoligotyping and 24-loci MIRU-VNTR typing results 38
4.1.2 Association of genotypes and drug resistance with treatment outcome 39
4.1.3 Association of genotypes with drug resistance and patient demographics 39
4.2 Discussion 44
4.3 Conclusion 47

CHAPTER 5 CONCLUDING REMARKS 49

5.1 Conclusions 49
5.2 Future research 50
5.3 Acknowledgements 51
References 52

Appendices 69
A: DETAILED METHODOLOGY
B: DETAILED RESULTS
C: STATISTICAL ANALYSIS
LIST OF TABLES PAGE

Table 2.1 Comparison of techniques for molecular typing or evaluating 29
genetic diversity within Mycobacterium tuberculosis
(Nicol and Wilkinson, 2008).

Table 4.3 The discriminatory power for M. tuberculosis of 41
Spoligotyping and MIRU-VNTR typing.

Table 4.4 Association of genotypes, drug resistance (Rifampicin and Isoniazid) 41
with the treatment outcome.

Table 4.5 Association between genotypes and gender. 42

Table 4.6 Association between genotype and age. 43

Table 4.7 Association between genotype and Isoniazid/Rifampicin drug resistance. 43

i
LIST OF FIGURES PAGE

Figure 2.1 The sanatorium where patients would spend months to years for the 6
treatment of tuberculosis during the 19th century.
Figure 2.2 Immune response to Mycobacterium tuberculosis infection. 9

Figure 2.3 Estimated new TB cases (WHO, 2013) 11

Figure 2.8 M. tuberculosis growing on LJ media 14

Figure 2.15 Molecular genotyping target sites on Mycobacterium tuberculosis 26
H37Rv, Structure of the DR locus in the mycobacterial genome of
Mycobacterium tuberculosis H37Rv and M. bovis BCG.

Figure 3.4 The different strains in Tshwane district area according to 39
spoligotyping.

Figure 4.1 A dendrogram for the combination for spoligotyping 40
and MIRU-VNTR

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