TABLE OF CONTENTS
ABSTRACT ii
ACKNOWLEDGEMENTS iv
DECLARATION v
APPROVAL SHEET vi
PERMISSION SHEET vii
TABLE OF CONTENTS viii
LIST OF TABLES xiii
LIST OF FIGURES xiv
LIST OF ABBREVIATIONS xv
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 6
2.1 Mycobacteria in General 6
2.1.1 The Genus Mycobacterium 6
2.1.2 Mycobacterium tuberculosis 7
2.2 Tuberculosis in General 8
2.2.1 Pathogenesis of Tuberculosis 8
2.2.2 Clinical Diagnosis of Tuberculosis 8
2.2.3 Treatment of Tuberculosis 9
2.2.4 Vaccination against Tuberculosis 10
2.2.5 Current Challenges for Tuberculosis 10
2.3 Expectoration of Mtb in Aerosol Droplet and Tuberculosis 11
Transmission
2.4 Laboratory Diagnosis of Tuberculosis 12
2.4.1 AFB Smear Microscopy 12
2.4.2 Mtb Isolation and Culture 13
2.4.3 Mycobacteriophage Amplification Assay 14
2.4.4 Nucleic Acid Amplification Techniques 15
2.4.4.1 PCR Detection 15
2.4.4.2 Loop-mediated Isothermal Amplification 16
Assay
2.5 Sampling and Quantification of Mtb in Respiratory 17
Droplets
2.5.1 Use of Animal Models 17
2.5.2 Air Filtration Coupled to PCR
2.5.3 Culture via the Cough Aerosol Sampling System 19
2.5.4 Use of Surgical Face Mask 21
3 METHODOLOGY 23
3.1 Apparatus and Consumables 23
3.2 Preparation of Culture Media 23
3.2.1 Middlebrook 7H9-OADC Broth and Agar 23
3.2.2 Middlebrook 7H9-OADC-Tween Broth 24
3.2.3 Middlebrook 7H9-OGC Broth and Agar 24
3.2.4 Middlebrook 7H10 Agar 24
3.2.5 Peptone Broth, 1% (w/v) with Tween 80, 1% (w/v) 24
3.2.6 Trypticase Soy Agar 25
3.3 Preparation of Reagents 25
3.3.1 Acid-alcohol 25
3.3.2 Calcein 25
3.3.3 Carbolfuchsin Stain 26
3.3.4 Ferrous Ammonium Sulfate, 200 mM 26
3.3.5 Glycerol Solution, 65% (v/v) 26
3.3.6 Methylene Blue Stain 26
3.3.7 Mycobacteriophage Buffer 27
3.3.8 NOA Antimicrobial Supplement 27
3.3.9 Tris-acetate-EDTA Buffer 27
3.3.10 Tween 80, 10% (w/v) 27
3.4 General Methods 28
3.4.1 Optical Density Measurement of Cultures 28
3.4.2 Cultivation of M. smegmatis 28
3.4.3 Cultivation of M. bovis BCG 29
3.4.4 Preparation of Glycerol Stock Cultures for Long- 29
term Storage
3.4.5 Preparation of M. smegmatis Culture Supernatant 29
3.4.6 Enumeration of Colony-forming Units 30
3.4.7 Ziehl-Neelsen Acid-fast Staining 30
3.5 Mycobacteriophage D29 31
3.5.1 Preparation of Phage Indicator Plate 31
3.5.2 Phage Propagation 31
3.5.3 Enumeration of Plaque-forming Units 31
3.6 Artificial Contamination of Face Masks with 32
Mycobacterial Cells
3.7 Direct Mask Analysis by the On-mask Phage Assay 37
3.8 Use of Mask Sampling to Study the Expectoration of 34
Respiratory Droplets by Healthy Subjects
3.8.1 Mask Sampling with Healthy Subjects 34
3.8.2 Processing of Worn Masks from Healthy 35
Subjects
3.8.3 NOA Decontamination of Worn Masks 35
3.9 Molecular Detection of Mycobacteria in Mask Samples 36
3.9.1 DNA Extraction from Mycobacteria in Mask 36
Eluate
3.9.2 Plaque DNA Extraction 36
3.9.3 Mtb complex-specific 16S rDNA LAMP Assay 37
3.9.4 IS1081 PCR Assay 38
3.9.5 Gel Analysis of PCR Amplicons 39
4 RESULTS 40
4.1 Evaluation of the Phage Assay for Direct Detection 40
of Mycobacterial Cells on Face Masks
4.1.1 Assessment of the On-mask Phage Assay for 41
Potential Occurrence of Background PFU Counts
4.1.2 Penetration of Spiked Mycobacterial Cells through 42
the Face Mask
4.1.3 Evaluation of the Sensitivity of the On-mask 43
Phage Assay with Artificially Spiked Mask Samples
4.1.4 Evaluation of NOA Decontamination of 44
Contaminated Masks Prior to the Phage Assay
4.1.5 Effect of Storage of Face Masks on the 46
Detection of Mycobacterial Cells by the
On-mask Phage Assay
4.2 Evaluation of the Feasibility of a Molecular Assay for 48
Detection of Target Mycobacterial Species in Phage D29
Plaques
4.2.1 Feasibility of the Mtb Complex-specific 16S 49
LAMP Assay
4.2.2 Feasibility of the Conventional IS1081 PCR Assay 50
4.2.3 Detection of Target Mycobacterial Species in 51
Phage D29 Plaques
4.3 Evaluation of the Sensitivity of Molecular Detection 53
of Mycobacterial Cells in Mask Eluates
4.4 Assessment of Levels and Distributions of Culturable 54
Respiratory Aerosols on Face Masks of Healthy
Subjects with Different Expiratory Maneuvers
5 DISCUSSION 58
5.1 Evaluation of the Phage Assay for Direct Detection 58
of Mycobacterial Cells on Face Masks
5.1.1 Assessment of the On-mask Phage Assay for 58
Potential Occurrence of Background PFU Counts
5.1.2 Penetration of Spiked Mycobacterial Cells through 59
the Face Mask
5.1.3 Evaluation of the Sensitivity of the On-mask 60
Phage Assay with Artificially Spiked Mask Samples
5.1.4 Evaluation of NOA Decontamination of 60
Contaminated Masks Prior to the Phage Assay
5.1.5 Effect of Storage of Face Masks on the 61
Detection of Mycobacterial Cells by the
On-mask Phage Assay
5.2 Evaluation of the Feasibility of a Molecular Assay for 63
Detection of Target Mycobacterial Species in Phage D29
Plaques
5.2.1 Feasibility of the Mtb Complex-specific 16S 63
LAMP Assay
5.2.2 Conventional IS1081 PCR Assay 64
5.2.3 Detection of Target Mycobacterial Species in 66
Phage D29 Plaques
5.3 Evaluation of the Sensitivity of Molecular Detection 67
of Mycobacterial Cells in Mask Eluates
5.4 Assessment of Levels and Distributions of Culturable 68
Respiratory Aerosols on Face Masks of Healthy
Subjects with Different Expiratory Maneuvers
5.4 Future Works 69
6 CONCLUSIONS 72
REFERENCES 75
APPENDIX 84
LIST OF TABLES
Table Page
3.1 Sequences of Mtb complex-specific 16S rDNA LAMP primers 37
3.2 Oligonucleotide primers used in the IS1081 PCR assay 38
3.3 Components of the IS1081 PCR assay 38
3.4 Profile for the IS1081 PCR assay 39
4.1 Assessment of the on-mask phage assay for the potential 41
occurrence of background PFUs
4.2 Penetration of spiked M. smegmatis cells through face masks 42
4.3 Evaluation of the sensitivity of the phage assay 43
4.4 Effect of NOA decontamination of contaminated face masks 45
from five healthy subjects
A1 List of apparatus and their manufacturers 84
A2 List of consumables and their manufacturers 85
LIST OF FIGURES
Figure Page
2.1 Schematic representation of LAMP primers 16
2.2 Cough aerosol sampling system showing a child coughing into 21
the sampling chamber through the inlet tubing
3.1 The commercial surgical face mask 33
3.2 On-mask phage infection of artificially contaminated mask 34
sample
4.1 Different plaque densities in the phage indicator plates 40
following overnight incubation at 37°C
4.2 Effect of storage of spiked mask segments on the detection of 46
M. smegmatis cells on them by the phage assay
4.3 Effect of treatment of stored mask segments 48
4.4 Visual assessment of Mtb complex-specific 16S LAMP 49
reactions via calcein fluorescence
4.5 Gel analysis of IS1081 PCR reactions in the assay specificity test 50
4.6 Gel analysis of M. bovis BCG IS1081 amplicons in the assay 51
sensitivity test
4.7 Gel analysis of IS1081 PCR reactions with extracts from 53
M. bovis BCG phage indicator plates
4.8 Distribution of CFUs on face masks of healthy subjects 56
4.9 Distribution of CFUs in the middle segment of face masks 57
of healthy subjects with different expiratory activities
5.1 Competitive inhibition exhibited by calcium ions to 67
inhibit DNA amplification during PCR
LIST OF ABBREVIATIONS
AFB acid-fast bacilli
BCG Bacille Calmette-Guerin
CaCl2 calcium chloride
CDC Centers of Disease Control and Prevention
CFU colony-forming unit
dNTP deoxyribonucleoside triphosphate
dH2O distilled water
DNA deoxyribonucleic acid
EDTA ethylenediaminetetraacetic acid
FAS ferrous ammonium sulfate
HCl hydrochloric acid
HIV human immunodeficiency virus
IS insertion sequence
LAMP loop-mediate isothermal amplification
M7H9 Middlebrook 7H9
M7H10 Middlebrook 7H10
MnCl2 Manganese chloride
MgSO4 magnesium sulfate
MP mycobacteriophage
Mtb Mycobacterium tuberculosis
NaCl sodium chloride
NTC no-template control
NTM non-tuberculous mycobacteria
OADC oleic acid-albumin-dextrose-catalase
OD optical density
PCR polymerase chain reaction
PFU plaque-forming unit
Rpf resuscitation-promoting factor
TAE Tris-acetate-EDTA
TB tuberculosis
TSA trypticase soy agar
UTAR Universiti Tunku Abdul Rahman
UV ultraviolet
WHO World Health Organization
bp base pair
cm centimeter
fg femtogram
g gram
M molar
rpm revolutions per minute
ml milliliter
mM millimolar
µl microliter
µM micromolar
ng nanogram
pg picogram
U enzyme unit
v/v volume per volume
w/v weight per volume
x g times gravity
.
