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
21 Mycobacteria in General 6
211 The Genus Mycobacterium 6
212 Mycobacterium tuberculosis 7
22 Tuberculosis in General 8
221 Pathogenesis of Tuberculosis 8
222 Clinical Diagnosis of Tuberculosis 8
223 Treatment of Tuberculosis 9
224 Vaccination against Tuberculosis 10
225 Current Challenges for Tuberculosis 10
23 Expectoration of Mtb in Aerosol Droplet and Tuberculosis 11
Transmission
24 Laboratory Diagnosis of Tuberculosis 12
241 AFB Smear Microscopy 12
242 Mtb Isolation and Culture 13
243 Mycobacteriophage Amplification Assay 14
244 Nucleic Acid Amplification Techniques 15
2441 PCR Detection 15
2442 Loop-mediated Isothermal Amplification 16
Assay
25 Sampling and Quantification of Mtb in Respiratory 17
Droplets
251 Use of Animal Models 17
252 Air Filtration Coupled to PCR
253 Culture via the Cough Aerosol Sampling System 19
254 Use of Surgical Face Mask 21
3 METHODOLOGY 23
31 Apparatus and Consumables 23
32 Preparation of Culture Media 23
321 Middlebrook 7H9-OADC Broth and Agar 23
322 Middlebrook 7H9-OADC-Tween Broth 24
323 Middlebrook 7H9-OGC Broth and Agar 24
32
4 Middlebrook 7H10 Agar 24
325 Peptone Broth, 1% (w/v) with Tween 80, 1% (w/v) 24
326 Trypticase Soy Agar 25
33 Preparation of Reagents 25
331 Acid-alcohol 25
332 Calcein 25
333 Carbolfuchsin Stain 26
334 Ferrous Ammonium Sulfate, 200 mM 26
335 Glycerol Solution, 65% (v/v) 26
336 Methylene Blue Stain 26
337 Mycobacteriophage Buffer 27
338 NOA Antimicrobial Supplement 27
339 Tris-acetate-EDTA Buffer 27
3310 Tween 80, 10% (w/v) 27
34 General Methods 28
341 Optical Density Measurement of Cultures 28
342 Cultivation of M smegmatis 28
343 Cultivation of M bovis BCG 29
344 Preparation of Glycerol Stock Cultures for Long- 29
term Storage
345 Preparation of M smegmatis Culture Supernatant 29
346 Enumeration of Colony-forming Units 30
347 Ziehl-Neelsen Acid-fast Staining 30
35 Mycobacteriophage D29 31
351 Preparation of Phage Indicator Plate 31
352 Phage Propagation 31
353 Enumeration of Plaque-forming Units 31
36 Artificial Contamination of Face Masks with 32
Mycobacterial Cells
37 Direct Mask Analysis by the On-mask Phage Assay 37
38 Use of Mask Sampling to Study the Expectoration of 34
Respiratory Droplets by Healthy Subjects
381 Mask Sampling with Healthy Subjects 34
382 Processing of Worn Masks from Healthy 35
Subjects
383 NOA Decontamination of Worn Masks 35
39 Molecular Detection of Mycobacteria in Mask Samples 36
391 DNA Extraction from Mycobacteria in Mask 36
Eluate
392 Plaque DNA Extraction 36
393 Mtb complex-specific 16S rDNA LAMP Assay 37
394 IS1081 PCR Assay 38
395 Gel Analysis of PCR Amplicons 39
4 RESULTS 40
41 Evaluation of the Phage Assay for Direct Detection 40
of Mycobacterial Cells on Face Masks
411 Assessment of the On-mask Phage Assay for 41
Potential Occurrence of Background PFU Counts
412 Penetration of Spiked Mycobacterial Cells through 42
the Face Mask
413 Evaluation of the Sensitivity of the On-mask 43
Phage Assay with Artificially Spiked Mask Samples
414 Evaluation of NOA Decontamination of 44
Contaminated Masks Prior to the Phage Assay
415 Effect of Storage of Face Masks on the 46
Detection of Mycobacterial Cells by the
On-mask Phage Assay
42 Evaluation of the Feasibility of a Molecular Assay for 48
Detection of Target Mycobacterial Species in Phage D29
Plaques
421 Feasibility of the Mtb Complex-specific 16S 49
LAMP Assay
422 Feasibility of the Conventional IS1081 PCR Assay 50
423 Detection of Target Mycobacterial Species in 51
Phage D29 Plaques
43 Evaluation of the Sensitivity of Molecular Detection 53
of Mycobacterial Cells in Mask Eluates
44 Assessment of Levels and Distributions of Culturable 54
Respiratory Aerosols on Face Masks of Healthy
Subjects with Different Expiratory Maneuvers
5 DISCUSSION 58
51 Evaluation of the Phage Assay for Direct Detection 58
of Mycobacterial Cells on Face Masks
511 Assessment of the On-mask Phage Assay for 58
Potential Occurrence of Background PFU Counts
512 Penetration of Spiked Mycobacterial Cells through 59
the Face Mask
513 Evaluation of the Sensitivity of the On-mask 60
Phage Assay with Artificially Spiked Mask Samples
514 Evaluation of NOA Decontamination of 60
Contaminated Masks Prior to the Phage Assay
515 Effect of Storage of Face Masks on the 61
Detection of Mycobacterial Cells by the
On-mask Phage Assay
52 Evaluation of the Feasibility of a Molecular Assay for 63
Detection of Target Mycobacterial Species in Phage D29
Plaques
521 Feasibility of the Mtb Complex-specific 16S 63
LAMP Assay
522 Conventional IS1081 PCR Assay 64
523 Detection of Target Mycobacterial Species in 66
Phage D29 Plaques
53 Evaluation of the Sensitivity of Molecular Detection 67
of Mycobacterial Cells in Mask Eluates
54 Assessment of Levels and Distributions of Culturable 68
Respiratory Aerosols on Face Masks of Healthy
Subjects with Different Expiratory Maneuvers
54 Future Works 69
6 CONCLUSIONS 72
REFERENCES 75
APPENDIX 84
LIST OF TABLES
Table Page
31 Sequences of Mtb complex-specific 16S rDNA LAMP primers 37
32 Oligonucleotide primers used in the IS1081 PCR assay 38
33 Components of the IS1081 PCR assay 38
34 Profile for the IS1081 PCR assay 39
41 Assessment of the on-mask phage assay for the potential 41
occurrence of background PFUs
42 Penetration of spiked M smegmatis cells through face masks 42
43 Evaluation of the sensitivity of the phage assay 43
44 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
21 Schematic representation of LAMP primers 16
22 Cough aerosol sampling system showing a child coughing into 21
the sampling chamber through the inlet tubing
31 The commercial surgical face mask 33
32 On-mask phage infection of artificially contaminated mask 34
sample
41 Different plaque densities in the phage indicator plates 40
following overnight incubation at 37°C
42 Effect of storage of spiked mask segments on the detection of 46
M smegmatis cells on them by the phage assay
43 Effect of treatment of stored mask segments 48
44 Visual assessment of Mtb complex-specific 16S LAMP 49
reactions via calcein fluorescence
45 Gel analysis of IS1081 PCR reactions in the assay specificity test 50
46 Gel analysis of M bovis BCG IS1081 amplicons in the assay 51
sensitivity test
47 Gel analysis of IS1081 PCR reactions with extracts from 53
M bovis BCG phage indicator plates
48 Distribution of CFUs on face masks of healthy subjects 56
49 Distribution of CFUs in the middle segment of face masks 57
of healthy subjects with different expiratory activities
51 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
