DEVELOPMENT OF A MASK SAMPLING METHOD FOR COLLECTION AND QUANTIFICATION OF MYCOBACTERIUM TUBERCULOSIS IN RESPIRATORY DROPLETS

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DEVELOPMENT OF A MASK SAMPLING METHOD FOR COLLECTION AND QUANTIFICATION OF MYCOBACTERIUM TUBERCULOSIS IN RESPIRATORY DROPLETS

By

TEE HAN KANG

Presented To

Department of Science Laboratory Technology

102 Pages 19,613 Words 1,737 Views PDF Format No. of Chapters: 1 to 6 Chapters

ABSTRACT

Mycobacterium tuberculosis (Mtb) is transmitted via the airborne route in the respiratory droplets expectorated by active pulmonary tuberculosis (TB) patients. To date, there is still lack of an established method for sampling respiratory aerosol droplets and measuring the Mtb concentration in them. Current medical practice continues to rely heavily on the acid-fast bacilli (AFB) smear status to gauge the infectiousness of TB patients. However, several limitations have been reported that challenge its reliability. In this project, a mask sampling approach was developed as the potential alternative to AFB smear microscopy for the assessment of TB infectivity. Mycobacterium smegmatis was used as the nonpathogenic surrogate model for Mtb throughout this study. Surgical masks were artificially spiked with M. smegmatis culture and then subjected to the on-mask phage assay. The detection limit observed was 100-1,000 CFUs, which is 104 to 105 folds higher than the method of PCR on mask eluates. In order to identify the target mycobacterial species in the resulting plaques, a PCR assay targeting the multicopy Mtb complex-specific IS1081 was performed downstream. However, it was unsuccessful in several trials, possibly due to inhibition by substances that were co-extracted with the target DNA from the phage plaque. Evaluation with masks worn by healthy subjects that were voluntarily coughed upon showed that the use of an antibiotic cocktail was efficient in completely preventing contamination of the phage assay by rapid-growing microbiota. Further studies with healthy volunteers also demonstrated that culturable respiratory aerosols were mainly sampled in the middle section of the mask and coughing, when compared to talking, yielded higher level of culturable aerosols on average. With further improvements and optimizations, the mask sampling approach could potentially be more reliable than AFB smear status in gauging TB infectiousness. This method could facilitate better understanding of TB transmission and provide a new platform for TB diagnosis.

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

102 Pages 19,613 Words 1,737 Views PDF Format No. of Chapters: 1 to 6 Chapters

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