DEVELOPl\1ENT AND EVALUATION OF A

NASBA SYSTEM FOR THE DIAGNOSIS OF CHOLERA

USING ELISA AND BIOSENSOR METHODS

by

LEESUYIN

Thesis submitted in fulfillment of the

requirements for the degree of

Doctor of Philosophy

April2008

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to my supervisor, Dr P. Lalitha, who

has been helpful and supportive throughout this study. Her critical comments and valuable

insights have guided me in the preparation of this thesis. I am grateful to my co-supervisor,.

Prof. Zainul F. Zainuddin and Assoc. Prof. M. Ravichandran for their guidance and advice

that have motivated me to become a better student and researcher.

To my dear friends and comrades, Melissa Chan, Yean Yean and Kurunathan, I

express my heartfelt appreciation for their encouragements and support that have helped me

through numerous failed experiments and mental roadblocks. My appreciation also goes out

to my labmates and friends who have helped me in one way or another during the course of

this study: Elina, Balqis, Donn, Chua, Siew Keah, Nik, Nurul, Kak Shikin, Nas, Nisha,

Chandrika, Syazwan, Kun Lee, Narjit and Kavitha. In addition, I am grateful to the lecturers,

administrative staff and lab technologists from the Department of Medical Microbiology and

Parasitology, and Institute for Research in Molecular Medicine, Universiti Sains Malaysia

for their help and the use of their excellent facilities.

I would like to thank Prof. Mitsuaki Nishibuchi from the Center for Southeast Asia

Studies, Kyoto University, Japan; Communicable Disease Hospital, Thandiarpettai, Chennai,

India; National Public Health Laboratory, Ministry of Health, Malaysia; Institute for Medical

Research (IMR), Malaysia and Kak Liza from the Medical Microbiology and Parasitology

laboratory, Universiti Sains Malaysia for providing the various bacterial strains used in this

study. I am also grateful to the Ministry of Science, Technology and Innovations (MOSTI)

for the IRPA and NBD grants that have funded part of this study.

On a more personal note, my family has been my strongest motivation and

inspirati6n. No words could ever express my gratitude to them. As a sign of appreciation, I

dedicate this thesis to them and hope that I have made them proud.

11

TABLE OF CONTENTS

Acknowledgement. ................................................................................................................... ii

Table of Contents .................................................................................................................... .iii

List ofTables ........................................................................................................................... .ix

List of Figures .......................................................................................................................... x

List of Symbols, Abbreviations and Acronyms ..................................................................... xiii

Abstrak ................................................................................................................................. xvii

Abstract. ................................................................................................................................. xix

CHAPTER 1 - INTRODUCTION

1.1 History of cholera ......................................................................................................... 1

1.2 Prevalence of cholera ................................................................................................... 1

1.3 Characteristics of V. cholerae ...................................................................................... 2

1.4 Ecology of V. cholerae ... .............................................................................................. 5

1.5 Viable but non-culturable (VBNC) state ...................................................................... 5

1.6 Epidemiology of cholera .............................................................................................. 6

1. 7 Pathophysiology of cholera .......................................................................................... 6

1.8 Clinical manifestations of cholera ................................................................................ 7

1.9 Treatment of cholera .................................................................................................... 8

1.10 Prevention of cholera ................................................................................................... 8

1.11 Diagnosis of cholera ..................................................................................................... 9

1.11.1 Conventional laboratory methods ................................................................... 9

1.11.2 Alternative methods of cholera diagnosis ..................................................... 11

1.11.2.1 Immunological-based tests for cholera .............................................. 11

1.11.2.2 Molecular-based tests for cholera ...................................................... 12

1.11.2.2.1 Characteristics and advantages of molecular-based tests

over conventional methods ........................................................ 13

1.11.2.3 Nucleic acid sequence-based amplification (NASBA) ....................... 14

1.12 Rationale of the study ................................................................................................. 17

1.13 Objectives of the study ............................................................................................... 18 I

1.14 Overview of the study ................................................................................................ 19

lll

CHAPTER 2- MATERIALS AND METHODS

2.1 Materials ..................................................................................................................... 20

2.1.1 Bacterial strains ............................................................................................. 20

2.1.1.1

2.1.1.2

2.1.1.3

2.1.2

2.1.3

2.1.3.1

2.1.3.2

2.1.4

2.1.5

2.1.6

2.1.6.1

2.1.6.2

Reference strains .................................................................................. 20

Clinical isolates .................................................................................... 20

Growth and maintenance of bacterial strains ........................................ 21

Culture Media ................................................................................................ 21

Primers and probes ........................................................................................ 25

Preparation of primer stock solutions ................................................... 25

Preparation of primer working solutions .............................................. 25

Deoxyribonucleotide triphosphates (dNTP, 20 !J.M) ..................................... 25

Ribonucleotides (NTP, 20 1-!M) ..................................................................... 26

Buffers and chemical stock solutions ............................................................ 26

Diethylpyrocarbonate (DE PC) treatment of water and solutions ......... 27

Diethylpyrocarbonate (DEPC) treatment of glasswares and

plasticwares .......................................................................................... 27

2.2 Methods ...................................................................................................................... 27

2.2.1 RNA Isolation ............................................................................................... 27

2.2.2 Quantitation of RNA ..................................................................................... 28

2.2.3 Plasmid Isolation ........................................................................................... 29

2.2.4 Genomic DNA Isolation ............................................................................... 29

2.2.5 Quantitation of DNA ..................................................................................... 30

2.2.6 Polymerase Chain Reaction (PCR) assay ................. , .................................... 31

2.2.7 Purification ofPCR product .......................................................................... 31

2.2.8 Agarose gel electrophoresis .......................................................................... 32

2.3 Experimental overview of Phase 1 study: Development and optimization of a

cholera NASBA assay ................................................................................................ 35

2.3.1 Designing primers and probes for cholera NASBA assay ........................... 36

2.3.2

2.3.2.1

2.3.2.2

2.3.2.3 i

2.3.2.4

2.3.2.5

2.3.2.6

2.3.2.7

Construction of in vitro RNA transcripts ...................................................... 38

Cloning of V. cholerae lo!B gene ......................................................... 38

PCR ofthe lolB gene ............................................................................ 38

Ligation ofPCR product into pTZS7R vector. ..................................... 38

Preparation of competent E. coli Top 10 cells ....................................... 40

Transforn1ation ..................................................................................... 40

PCR screening of transformed colonies ............................................... 41

DNA sequencing .................................................................................. 43

IV

2.3.2.8

2.3.2.9

2.3.2.10

Plasmid linearization by restriction endonuclease digestion ................ 43

In vitro transcription ............................................................................. 43

Purification of RNA using phenol-chloroform and ethanol

precipitation .......................................................................................... 44

2.3.3 Cholera NASBA assay ................................................................................. 46

2.3.3.1

2.3.3.2

Preparation of cholera NASBA reaction mix and enzyme mix ............ 46

Detection ofNASBA amplicons by agarose gel electrophoresis ......... 49

2.3 .3 .3 Optimization of cholera NASBA reaction conditions .......................... 49

2.3.3.4 Analytical specificity of the cholera NASBA assay ............................. 51

2.3.3.5 Analytical sensitivity of the cholera NASBA assay ............................. 51

2.3.3.5.1 Analytical sensitivity at the gene level ........................................ 51

2.3.3.5.2

2.3.3.6

Analytical sensitivity at the bacterial cell level ........................... 52

Reproducibility and efficiency of the RNA isolation technique .......... 53

2.3.4

2.3.4.1

Analysis of the NASBA reaction and its amplicons ..................................... 54

Northern Blotting .................................................................................. 54

2.3.4.2 Dot-blotting .......................................................................................... 55

2.3.4.2.1 Hybridization with biotinylated DNA probe ................................ 55

2.3.4.2.2 Hybridization with Streptavidin-HRP ........................................... 56

2.3.4.2.3 Colorimetric detection ofblots ..................................................... 56

2.3.4.3 RNase and DNase digestion ofNASBA amplicons ............................. 57

2.3.4.4 Amplification of DNA by NASBA ...................................................... 59

2.3.4.5 Viability assay ...................................................................................... 59

2.4 Experimental overview of Phase 2 study : Development and optimization of

ELISA for detection ofNASBA amplicons (NASBA-ELISA) ................................. 61

2.4.1 Cholera NASBA-ELISA assay ..................................................................... 62

2.4.1.1 Preparation of 3 '-end biotin-tailed capture probe

2.4.1.2

2.4.1.3

2.4.1.4

2.4.1.5

2.4.1.6

2.4.1.7

(VHA-S4-Biotin3 ') ............................................................................... 64

Optimization and comparison of the hybridization efficiency of

3 '-end biotin-tailed capture probe (VHA-S4-Biotin3 ')and

5' -end biotin- labelled capture probe (VHA-S4-5'Biotin) ................... 64

Optimization ofVN2F-Flu concentration as detection probe for

unlabelled NASBA amplicons .............................................................. 65

Optimization of fluorescein-12-UTP concentration for internal

labeling ofNASBA amp1icons ............................................................. 65

Detection ofNASBA amplicons by ELISA ......................................... 65

Analytical specificity of the cholera NASBA-ELISA assays ............... 67

Analytical sensitivity of the cholera NASBA-ELISA assays ............... 67

v

2.4.2

2.4.2.1

2.4.2.2

Clinical evaluation of cholera NASBA-ELISA using spiked stool

samples ......................................................................................................... 68

Preparation of spiked stool samples ..................................................... 68

Limit of detection (LoD) of cholera NASBA-ELISA at the

bacterial cell level using spiked stool samples ..................................... 69

2.4.2.3 Statistical analysis ................................................................................ 69

2.4.2.3.1 Calculation of sample size for spiked stool sample evaluation .... 69

2.4.3.3.2 Calculation of diagnostic performance for spiked stool

sample evaluation ......................................................................... 70

2.5 Experimental overview of Phase 3 study: Development of electrochemical

biosensor detection for cholera NASBA-ELISA ....................................................... 71

2.5.1 Biosensor detection for cholera NASBA-ELISA .......................................... 72

2.5.1.1 Biosensor and electrode design ............................................................ 72

2.5.1.2 Optimization of biosensor detection of cholera NASBA-ELISA

signal .................................................................................................... 72

2.5.1.3 Analytical specificity of the cholera NASBA-ELISA with biosensor

detection ............................................................................................... 73

2.5.1.4 Analytical sensitivity of the cholera NASBA-ELISA with biosensor

detection ................................................................................................ 73

2.6 Experimental overview of Phase 4 study: Optimization of a thermostabilized

cholera NASBA mix .................................................................................................. 75

2.6.1 Thermostabilization of cholera NASBA mix (reaction mix and

enzyme mix) .................................................................................................. 76

2.6.1.1 Preparation offreeze-dried reaction mix .............................................. 76

2.6.1.2 Preparation of freeze-dried enzyme mix .............................................. 77

2.6.1.2.1 Optimization of enzymes and enzyme stabilizer concentrations ... 77

2.6.1.3 Freeze-drying ofNASBA reaction and enzyme mixes ........................ 77

2.6.1.4 Reconstitution ofthe freeze-dried NASBA mix.. ................................. 78

2.6.1.5 Analytical sensitivity of the freeze-dried cholera NASBA mix at

the bacterial cell level.. ......................................................................... 78

2.6.1.6 Determination of periodic stability of the freeze-dried cholera

NASBA mix at different temperatures ................................................. 79

vi

CHAPTER 3- RESULTS AND DISCUSSION ·

3.1 Designing primers and probes for cholera NASBA assay ......................................... 80

3.2 Construction of RNA transcript ................................................................................. 84

3.2.1 Cloning of lolB gene into pTZ57R vector .................................................... 84

3.2.2 Confirmation of clones by PCR screening and DNA sequencing ................. 86

3.2.3 In vitro transcription of the linearized pTZ-lolB plasmid ............................. 89

3.3 Cholera NASBA assay ............................................................................................... 91

3.3.1 Optimization of cholera NASBA reaction conditions ................................... 91

3.3.1.1 MgCh concentration ............................................................................. 91

3.3.1.2 KCl concentration ................................................................................. 91

3.3.1.3

3.3.1.4

3.3.1.5

3.3.2

3.3.3

3.3.4

3.3.5

Primers concentration ........................................................................... 93

DMSO concentration ............................................................................ 93

Enzymes concentration ......................................................................... 95

Detection ofNASBA amplicons by agarose gel electrophoresis .................. 98

Analytical specificity of the cholera NASBA assay ..................................... 98

Analytical sensitivity ofthe cholera NASBA assay .................................... 100

Reproducibility and efficiency of the RNA isolation technique ................. 103

3.3.6 Analysis of the NASBA reaction and its amplicons ................................... 105

3.3.6.1 Probe-based hybridization ofNASBA amplicons .............................. 105

3.3.6.1.1 Northern Blotting ........................................................................ 105

3.3.6.1.2

3.3.6.2

3.3.6.3

Dot-blotting ................................................................................. 105

RNase and DNase digestion ofNASBA amplicons ........................... 107

Amplification of DNA by NASBA .................................................... 109

3.3.6.4 Viability assay .................................................................................... 111

3.4 Cholera NASBA-ELISA .......................................................................................... 113

3.4.1 Optimization and comparison of 3 '-end biotin-tailed capture probe

(VHA -S4-Biotin3 ') and 5 '-end biotin-labelled capture probes

(VHA-S4-'Biotin) ....................................................................................... 113

3.4.2 Optimization ofVN2F-Flu concentration as detection probe for

unlabelled NASBA amplicons .................................................................... 115

3.4.3 Optimization of fluorescein-12-UTP concentration for internal labeling

ofNASBA amplicons ................................................................................. 117

3.4.5 Analytical specificity of the two NABSA-ELISA formats ......................... 119

3.4.6 Analytical sensitivity of the two NASBA-ELISA formats at the gene

and bacterial cell level.. ............................................................................... 122

3.4.7 Comparison of performance ofthe two NASBA-ELISA formats .............. 126

Vll

3.4.8

3.4.8.1

3.4.8.2

Evaluation of cholera NASBA-ELISA using spiked stool samples ............ 128

Diagnostic performance of the cholera NASBA-ELISA assay .......... 131

Limit of detection (LoD) of cholera NASBA-ELISA at the

bacterial cell level using spiked stool samples ................................... 131

3.5 Electrochemical biosensor detection of cholera NASBA-ELISA ........................... 134

3.5.1 Analytical specificity ofthe cholera NASBA-ELISA assay with

biosensor detection ...................................................................................... 134

3.5.2 Analytical sensitivity of the cholera NASBA-ELISA assay with

biosensor detection ...................................................................................... 137

3.5.3 Comparison and correlation between biosensor- and spectrophotometric-

based ELISA results .................................................................................... 139

3.6 Thermostabilization of cholera NASBA mix .......................................................... 141

3.6.1 Optimization of enzymes and enzyme stabilizer concentrations ................ 141

3.6.2 Analytical sensitivity of the freeze-dried NASBA mix at the bacterial

cell and gene level ....................................................................................... 144

3.6.3 Periodic stability of the freeze-dried NASBA mix ..................................... 146

3.7 Discussion ................................................................................................................ 148

CHAPTER 4- SUMMARY AND CONCLUSION ......................................................... 167

REFERENCES ..................................................................................................................... 170

APPENDICES

Appendix A- Preparation of Culture Media, Buffers and Chemical Stock

Solutions .......................................................................................................................... A 1

Appendix B- Alignment result of V cholerae lo!B gene (AF227752) with the

lo!B or hemM gene of other enteric bacteria .................................................................. All

Appendix C -lo!B (hemM) sequence (Genbank accession no: AF227752) and

NASBA primers and probes binding sites ...................................................................... Al6

Appendix D- Partial sequence of pTZ-lolB plasmid (position 551 - 1350)

showing the binding sites of primers used for PCR screening of clones and restriction

enzymes sites .................................................................................................................. A 17 I

Appendix E- Alignment of pTZ-lolB sequencing result with the /alB gene

sequence (AF227752) ..................................................................................................... Al8

LIST OF PUBLICATIONS

Vlll

LIST OF TABLES

Table 1.1: Tests for differentiation of Classical and El Tor biotypes of V. cholerae

serogroup 0 1 .......................................................................................................... 4

Table 1.2: V. cholerae serogroups and their antigenic determinants ...................................... .4

Table 2.1: Details of the reference strains used in this study ................................................ 22

Table 2.2: Details of the clinical isolates used in this study ................. .-................................ 23

Table 2.3: Composition of a standard PCR mix .................................................................... 34

Table 2.4: Standard thermal cycling programme .................................................................. 34

Table 2.6: Details of the cholera NASBA primers and probes used in this study ................ 37

Table 2.5: Details ofPCR primers, annealing temperatures and expected product sizes

used in the PCR screening of transformed colonies ............................................. 42

Table 2. 7: Composition of the cholera NASBA mix before optimization ............................ 48

Table 2.8: Optimization of cholera NASBA reaction components ....................................... 50

Table 3.1: Result of PCR screening for two positive pTZ-lolB clones to confirm the

gene insert size and orientation ............................................................................ 88

Table 3.2: Composition of the optimized cholera NASBA mix ........................................... 97

Table 3.3: Reproducibility of the RNA isolation technique. RNA isolation was

performed on a culture of V. cholerae 01 El Tor in six replicates ..................... 104

Table 3.4: Efficiency of the RNA isolation technique. RNA isolation was performed

on four replicate samples spiked with 10 Jlg of RNA from V. cholerae

01 El Tor ............................................................................................................ 104

Table 3.5: Reproducibility ofNASBA-ELISA using unlabelled and labelled NASBA

amplicons based on the analytical sensitivity assays .......................................... 127

Table 3.6: Summary of cholera NASBA-ELISA evaluation with spiked stool samples .... 130

Table 3.7: Statistical analysis ofthe diagnostic performance of the cholera

NASBA-ELISA ................................................................................................. 132

ix

LIST OF FIGURES

Figure 2.1: Procedures involved in the cloning of lolB gene of V. cholerae ........................ 39

Figure 2.2: Plasmid map ofpTZ-lolB showing the exact position of the cloning site,

genetic elements, restriction sites and T7 promoter site ..................................... 39

Figure 2.3: In vitro transcription of the Sad linearized pTZ-lolB plasmid to produce

RNA transcripts ................................................................................................... 45

Figure 2.4: Procedures involved in NASBA and detection of its amplicons by agarose

gel electrophoresis ............................................................................................... 4 7

Figure 2.5: Procedures involved in the viability assay to determine whether the lolB

mRNA can be used as an indicator of cell viability ............................................ 60

Figure 2.6: Diagrammatic representation of the two ELISA formats evaluated in this

study using unlabelled NASBA amplicons (A) or fluorescein-labelled

NASBA amplicons (B) ........................................................................................ 63

Figure 2.7: Experimental overview ofthe cholera NASBA-ELISA assay with biosensor

detection ............................................................................................................. 74

Figure 3.1: Mfold result showing a possible structure for the 784 nucleotide region of

the lolB mRNA molecule and the NASBA primers binding sites ....................... 82

Figure 3.2: Mfold result showing a possible structure of the 203 nucleotide NASBA

amplicon and probes binding sites ...................................................................... 83

Figure 3.3: Agarose gel electrophoresis result of the lolB gene PCR product for cloning

into pTZS7R vector ............................................................................................. 85

Figure 3.4: PCR screening of two positive clones for the correct lolB gene insert size

and orientation ..................................................................................................... 87

Figure 3.5: Linearization ofpTZ-lolB plasmid with Sad enzyme ........................................ 90

Figure 3.6: In vitro transcription oflinearized pTZ-lolB plasmid ......................................... 90

Figure 3. 7: Optimization ofMgCh concentration ................................................................. 92

Figure 3.8: Optimization ofKCl concentration ..................................................................... 92

Figure 3.9: Optimization of primers concentration ............................................................... 94

Figure 3.10: Optimization ofDMSO concentration .............................................................. 94

Figure 3.11: Optimization ofT7 RNA polymerase (A) and AMV-RT (B) enzymes

concentration ..................................................................................................... 96

Figure 3.12: Analytical specificity evaluation of the cholera NASBA assay with

agarose gel electrophoresis detection ................................................................ 99

Figure 3.13: Analytical sensitivity of the cholera NASBA assay at the gene level using

agarose gel electrophoresis detection .............................................................. 101

X

Figure 3.14: Analytical sensitivity ofthe cholera NASBA assay at the bacterial cell

level using agarose gel electrophoresis detection ............................................ 102

Figure 3.15: Northern blotting ofNASBA amp1icons from agarose gel (A) onto a

positively-charged nylon membrane (B) ......................................................... I 06

Figure 3.16: Dot-blotting ofNASBA amplicons onto a positively-charged nylon

membrane ........................................................................................................ 106

Figure 3.17: Analysis ofNASBA amplicons by RNase A and DNase I digestion ............. 108

Figure 3.18: Analysis ofRNase A and DNase I digested NASBA amplicons by

RT-PCR (A) and PCR (B) ............................................................................... 108

Figure 3.I9: Amplification of DNA by NASBA ................................................................. II 0

Figure 3.20: Evaluation of the stability of the /olB mRNA after lethal treatments and

incubation at different times and temperatures .............................................. 112

Figure 3.21: Comparison ofELISA signal using 3'-end (VHA-S4-Biotin3') and 5'-end

(VHA-S4-5'Biotin) biotin-labelled capture probes ......................................... 114

Figure 3.22: Optimization ofVN2F-Flu detection probe concentration using unlabelled

NASBA ampiicons from positive control. ...................................................... 116

Figure 3.23: Optimization offluorescein-12-UTP concentration using /olB RNA transcripts

with amplicon detection by agarose gel electrophoresis(A) and ELISA(B) .... Il8

Figure 3.24: Analytical specificity of ELISA detection using unlabelled NASBA amplicons

evaluated using a panel of 41 reference bacterial strains ............................... 120

Figure 3.25: Analytical specificity of ELISA detection using fluorescein-labelled NASBA

amplicons evaluated using a panel of 41 reference bacterial strains ............ 121

Figure 3.26: Analytical sensitivity ofNASBA-ELISA at the (A) gene and (B) bacterial cell

level using unlabelled and fluorescein-labelled NASBA amplicons ......... 123

Figure 3.27: Analytical sensitivity at the gene level using unlabelled (A) and fluorescein-

labelled (B) NASBA amplicons by agarose gel electrophoresis ..................... 124

Figure 3.28: Analytical sensitivity at the bacterial cell level using unlabelled (A) and

fluorescein-labelled (B) NASBA amplicons by agarose gel electrophoresis .. .l25

Figure 3.29: Clinical evaluation of cholera NASBA-ELISA using spiked stool samples .. 129

Figure 3.30: Limit of detection (LoD) of cholera NASBA-ELISA at the bacterial cell

level with spiked stool samples ....................................................................... 133

Figure 3.31: Analytical specificity of cholera NASBA-ELISA with signal measurement by 1 spectrophotometry (microplate reader) and biosensor. ................................... 136

Figure 3.32: Analytical sensitivity ofNASBA-ELISA at the gene (A) and bacterial

cell (B) level with signal measurement by spectrophotometry (microplate

reader) and biosensor ....................................................................................... 138

XI

Figure 3.33: Correlation analysis of biosensor and spectrophotometry results ................... 140

Figure 3.34: First round optimization of enzymes and enzyme stabilizer concentrations

for thermostabilization of enzyme mix ............................................................ 142

Figure 3.35: Second round optimization of enzymes and enzyme stabilizer concentrations

for thermostabilization of enzyme mix ............................................................ 142

Figure 3.36: Optimization using higher concentrations of enzyme stabilizer for

thermostabilization of enzyme mix ................................................................. 143

Figure 3.37: Analytical sensitivity of the freeze-dried NASBA mix at the bacterial

cell level (A) and gene level (8) ...................................................................... 145

Figure 3.38: Periodic evaluation of the stability of freeze-dried NASBA mix .................... 147

xii

LIST OF SYMBOLS, ABBREVIATIONS AND ACRONYMS

nil eDNA complimentary deoxyribonucleic acid

% percent CPU colony forming unit

approximately cr chlorine ion

+ positive em centimeter

negative/ minus CT cholera toxin

< less than CTP cytidine 5 '-triphosphate

> more than cv coefficient of variation

± plus I minus dATP deoxyadenosine triphosphate

~ to dCTP deoxycytidine triphosphate

oc degree Celsius ddt dideoxythymidine

A adenine I adenosine; DEPC diethylpyrocarbonate absorbance

A26o absorbance at 260 nm dG Gibbs free energy

A2so absorbance at 280 nm dGTP deoxyguanosine triphosphate

A4so absorbance at 450 nm dH20 distilled water

ADP adenosine diphosphate DMSO dimethyl sulfoxide

Ag/AgCl silver/silver chloride DNA deoxyribonucleic acid

AMV-RT avian myeloblastosis virus- DNase deoxyribonuclease reverse transcriptase

APW alkaline peptone water dNTP deoxynucleoside triphosphate

ATP adenosine 5 '-triphosphate DTT dithiothreitol

BLAST Basic Local Alignment dTTP deoxythymidine triphosphate Search Tool

bp basepair ECL electrochemiluminescence

BSA bovine serum albumin EDTA ethylenediaminetetraacetic acid

c cytosine; current ELGA enzyme-linked gel assay

cAMP adenosine 3 ',5 '-cyclic ELISA enzyme-linked monophosphate immunosorbent assay

xiii

eta/. et alii I and other IU infectious unit; international unit

FAM carboxyfluorescein J Joule

FDA Food and Drug k kappa value Administration

Flu fluorescein K+ potassium ion

G guanine I guanosine kcallmol kilocalories per mol

g gram KCl potassium chloride

GHKB General Hospital Kota Bharu kDa kilodalton

GITC guanidine thiocyanate KIA Kligler iron agar

GM1 monosialotetrahexosylganglio L/hour liter per hour side

GMO genetically modified LB Luria Bertani organism

GTP guanosine 5 '-triphosphate LCD liquid crystal display

H2 hydrogen LCR ligase chain reaction

H20 water LoD limit of detection

H202 hydrogen peroxidase M molar I molarity

H2S hydrogen sulphate mbar millibar

HCl hydrogen chloride MCS multiple cloning site

HC03- hydrogen carbonate ion mg milligram

HIV-1 human immunodeficiency MgCh magnesium chloride virus 1

HPLC high performance liquid mm minute chromatography

hr hour MKAK National Public Health Laboratory

HRP horseradish peroxidase ml milliliter

HUSM Hospital Universiti Sains mm millimeter Malaysia

I.e. id est I that is mM millimolar

IFU 1 inclusion-forming unit M-MLV Moloney murine leukemia RT virus reverse transcriptase

IMR Institute for Medical MOPS 3-(N-Research morpholino )propanesulfonic

IPA intermittent pulse mRNA messenger ribonucleic acid amperomehy

XIV

N normal psi pounds per square inch

n sample size Psn specificity

N/A not available I not applicable Psp sensitivity

Na+ sodium ion R correlation coefficient

NAO nicotinamide red reduced I reduction

NaOH sodium hydroxide RNA ribonucleic acid

NASBA nucleic acid sequence-based RNase ribonuclease amplification

NCBI National Center for RNase H Ribonuclease H Biotechnology Information

nm nanometer RNasin RNasin Ribonuclease Inhibitor

nmol nanomol rpm revolutions per minute

NPV negative predictive value rRNA ribosomal ribonucleic acid

nt nucleotide RSO relative standard deviation

NTP nucleoside triphosphate RT room temperature

Oz oxygen RT-PCR reverse transcription-polymerase chain reaction

00 optical density s Svedberg unit

00450 optical density at 450 nm SAM salf-assembled monolayer

00600 optical density at 600 nm SO standard deviation

OH hydroxyl group SOA strand displacement amplification

ORS oral rehydration solution SOS sodium dodecyl sulphate

ox oxidized I oxidation s second

PBS phosphate buffered saline SPCE screen-printed carbon electrode

PBST phosphate buffered saline- SPE screen-printed electrode Tween 20

PCR polymerase chain reaction spp. species

PFU PCR-forming unit SPR surface plasmon resonance

pmol picomole sse sodium chloride/sodium citrate

PPV positive predictive value SSCT sodium chloride/sodium citrate/Tween 20

XV

T thymine I thymidine UTP uri dine 5 '-triphosphate

Ta annealing temperature uv ultraviolet

TAE Tris-acetate-EDT A v volts

TBE Tris-borate-EDTA v/v volume/volume

TCBS thiosulphate citrate bilesalt VBNC viable but non-culturable sucrose

tcp toxin coregulated pili vs versus

TdT terminal deoxynucleotidyl w/v weight/volume transferase

TE Tris-EDTA www World Wide Web

Tm melting temperature X precision

TMA transcription-mediated xg relative centrifugal force amplification

TMB 3,3',5,5'- JlA microamperes tetramethylbenzidine

Tris tris(hydroxymethyl)aminomet jlg microgram hane

tRNA transfer ribonucleic acid jll microliter

TSI triple sugar indole jlm micrometer

u units J.!M micromolar

xvi

PEMBANGUNAN DAN PENILAIAN SISTEM NASBA UNTUK DIAGNOSIS

PENYAKIT TAUN MENGGUNAKAN KAEDAH ELISA DAN BIOSENSOR

ABSTRAK

Taun (kolera) ialah penyakit ciri-birit yang disebabkan Vibrio cholerae. Taun boleh

membawa maut jika tidak dikesan dengan serta-merta. Justeru, pengesanan awal adalah

penting untuk rawatan pesakit dan kawalan wabak taun. Ujian kultur dan biokimia

konvensional adalah rumit, lambat dan kurang sensitif. Walaupun ujian berasaskan teknik

molekul adalah lebih pantas, sensitif dan spesifik, kaedah ini memerlukan peralatan mahal

dan penyimpanan sejuk bahan-bahan reagen. Ujian DNA seperti PCR tidak dapat

membezakan sel hidup dengan sel mati. Kaedah amplifikasi berasaskan asid nukleik

(NASBA) yang menggandakan RNA secara isoterma dapat mengesan sel hidup sahaja.

Maka, objektif kajian ini adalah untuk membangunkan dan menilai suatu ujian NASBA­

ELISA yang stabil haba dengan biosensor untuk mengesan V. cholerae berasaskan gen lolB.

Pada peringkat permulaan, transkrip RNA sebagai kawalan positif dikonstruk dan primer

serta prob khusus direka. Pengoptimuman ujian NASBA dan ELISA dilakukan dan

spesifisiti analitikal diuji dengan 41 bakteria rujukan yang terdiri daripada V. cholerae,

Vibrio spesis dan patogen enterik. Sensitiviti analitikal diuji dengan cairan bersiri transkrip

RNA dan sel-sel V. cholerae. Penilaian klinikal dijalankan dengan menggunakan najis yang

ditokok sampel (n=200). Seterusnya, kaedah biosensor dioptimurnkan dan keputusannya

dibandingkan dengan kaedah spektrofotometri. Campuran NASBA dijadikan stabil haba

melalui pengeringbekuan dan kestabilannya dinilai pada suhu berlainan untuk jangkamasa

tertentu. Selain itu, kesesuaian lolB mRNA sebagai penunjuk kebolehhidupan diselidiki

dengan rhembunuh sel-sel V. cholerae melalui pelbagai kaedah dan mengesan isyarat

NASBA daripadanya. Ujian kolera NASBA-ELISA yang telah dioptimurnkan berdasarkan

pengesanan produk amplifikasi melalui prob berlabel fluorescein dan isyarat TMB/HRP.

· Spesifisiti analitikal ujian adalah l 00% manakala sensitiviti analitikal ujian adalah 102

xvn

molekul/J..Ll transkrip RNA dan 10 CFU/ml sel. Penilaian klinikal memberikan keputusan

100% sensitif, 84.52% spesifik, 89.92% nilai ramalan positif dan 100% nilai ramalan negatif.

Kaedah biosensor didapati setanding kaedah spektrofotometri dengan mencatatkan kepekaan

analitikal yang sama, korelasi yang rapat (R2<0.964) dan persetujuan kappa yang hampir

sempuma (95.1%, k = 0.828). Penstabilan campuran NASBA mampu mengekalkan

kestabilannya pada 8°C dan -20°C selama dua bulan. Dalam ujian kebolehhidupan sel, gen

lolB masih dapat dikesan daripada kultur sel V. cholerae yang telah dibunuh selepas 48 jam,

justeru gen ini tidak sesuai dijadikan penunjuk sel bemyawa. Kesimpulannya, buat pertama

kalinya, kami telah membangunkan suatu ujian kolera-NASBA-ELISA dengan pengesanan

biosensor yang sensitif dan dapat dilakukan dengan peralatan mudah dalam empat jam.

Campuran NASBA kering mengurangkan langkah-langkah mempipet serta memudahkan

penghantaran dan penyimpanan ujian. Ujian ini sesuai sebagai ujian diagnostik pantas dan

ujian saringan di lapangan.

xviii

DEVELOPMENT AND EVALUATION OF A NASBA SYSTEM FOR THE

DIAGNOSIS OF CHOLERA USING ELISA AND BIOSENSOR METHODS

ABSTRACT

Cholera is a diarrheal disease caused by Vibrio cholerae. Cholera is potentially

lethal if not diagnosed on time. Hence, early detection is crucial for patient treatment and

containment of outbreak. Conventional culture and biochemical tests are laborious, time­

consuming and less sensitive. Although molecular-based methods are rapid, more

sensitive and specific; they require expensive equipments and cold storage of reagents.

Furthermore, DNA-based tests such as PCR, do not distinguish between viable and non­

viable cells. Nucleic acid sequence-based amplification (NASBA) is an isothermal RNA

amplification technique that specifically detects viable cells. Hence, the objective of this

study was to develop and evaluate a thermostabilized cholera-NASBA-ELISA assay with

biosensor detection for V. cholerae based on the lo/B gene. RNA transcripts as positive

control were first constructed and specific primers and probes were designed. NASBA and

ELISA conditions were optimized and the analytical specificity was tested with 41

reference strains comprising of V. cholerae, Vibrio species and enteric pathogens. The

analytical sensitivity was tested with serial dilutions of RNA transcripts and V. cholerae

cells. Clinical evaluation of the assay was performed using spiked stool samples (n=200).

Subsequently, biosensor detection for the NASBA-ELISA assay was optimized and the

results compared to spectrophotometry. The NASBA mix was thermostabilized by freeze­

drying and its stability at different temperatures was determined periodically. In addition,

suitability of lo/B mRNA as a viability indicator was investigated by subjecting cultures to

lethal tl1eatments and detecting the NASBA signal. The optimized cholera-NASBA­

ELISA assay detected amplicons using fluorescein-labeled probes and TMB/HRP signal.

The analytical specificity of the assay was 100%, while the analytical sensitivity was 102

molecules/Ill RNA transcripts and 10 CFU/ml cells. Clinical evaluation gave 100%

XIX

sensitivity, 84.52% specificity, 89.92% PPV and 100% NPV. Biosensor detection was

comparable to spectrophotometry, yielding similar analytical sensitivity level, excellent

correlation (R2 <0.964) and near perfect kappa agreement (95.1 %, k= 0.828).

Thermostabilization of the NASBA mix was able to preserve its stability at 8°C and -20°C

for two months. In the viability assay, lolB mRNA was detected even after 48 hours post­

treatment, therefore precluding its use as a viability indicator. In conclusion, we have for

the first time, developed a sensitive cholera-NASBA-ELISA assay with biosensor

detection that can be performed using simple equipments within four hours. The dry

NASBA mix reduces multiple pipetting steps and facilitates transportation and storage.

The test is suitable for use as a rapid diagnostic test or screening test in the field.

XX

1.1 History of cholera

CHAPTER 1

INTRODUCTION

Cholera is a potentially life-threatening secretory diarrheal disease caused by the

bacteria Vibrio cholerae. Cholera is thought to originate from the Ganges Delta of the Indian

subcontinent. In the nineteenth century, seven distinct pandemic waves of cholera spread

from Asia to many parts of the world. Six pandemics arose from the Indian subcontinent,

while the seventh pandemic originated from the island of Sulawesi, Indonesia in 1961 (Parsi,

2001). In the late 1992, a possible eighth pandemic was reported when a large epidemic

cholera spread throughout Bangladesh, India, Pakistan, Nepal, China, Thailand and Malaysia

(Blake, 1994).

There are more than 150 serogroups of V. cholerae, however only the serogroups 01

and 0139 cause epidemic and pandemic diseases (Sack et al., 2004). The Classical biotype

of V. cholerae serogroup 01 was responsible for the fifth and sixth pandemics and is

believed to have been associated with the earlier pandemics as well. The seventh cholera

pandemic is caused by the El Tor biotype of V. cholerae serogroup 01 and this biotype has

completely displaced the Classical biotype worldwide, except in Bangladesh where it

appeared in epidemic proportions in 1982 and now seems to have become extinct again

(WHO, 2002). The newly emerged serogroup 0139 or 'Bengal' in the late 1992 has been

attributed to the eighth cholera pandemic and this serogroup is closely related to the Asian

V. cholerae 01 El Tor strains associated with the seventh pandemic (Blake, 1994).

1.2 Prevalence of cholera

Cholera is one of the oldest and best-known diseases in the world, yet it continues to

cause considerable suffering and needless deaths. In 2006 alone 131,943 cases of cholera

were reported worldwide with 2272 fatalities (WHO, 2007). However, this number is just the

tip of the iceberg because poor surveillance and fear of travel and economic sanctions lead to

under-reporting of actual cholera cases in certain countries (WHO, 2007). It is estimated that

in reality, the number of deaths from cholera is probably 100 times more than was officially

reported (WHO, 2007). In Malaysia, the Ministry of Health reported the incidence rate of

cholera in 2005 (per 100,000 population) was 1.48 with a 0.01 mortality rate (Ministry of

Health Malaysia, July 2007).

1.3 Characteristics of V. cholerae

Vibrio cholerae is a member of the family Vibrionaceae. It is a facultative anaerobe,

short, curved rod about 1.4 - 2.6 f.!m, capable of fermentative and respiratory metabolism. It

is a non-halophilic vibrio, oxidase-positive, reduces nitrate and is highly motile by means of

a single sheathed polar flagellum. Even though growth of V. cholerae is stimulated by

addition of 1% sodium chloride, it can grow in nutrient broth without added sodium chloride,

which is an important distinction from other Vibrio spp. (Kay et al., 1994). It can grow at 16

- 40°C, with an optimum temperature of37°C and at pH 7.4-9.6 (WHO, 2002).

The species V. cholerae is divided into more than 150 serogroups based on the

differences in their somatic (0) antigens. These variations are secondary to differences in the

oligosaccharide side chains in parts of the cell wall (Parsi, 2001). The serogroup 01 is

divided into two biotypes, Classical and El Tor, which can be differentiated by tests such as

hemolysis, hemagglutination, phage typing, polymyxin B sensitivity and Voges-Proskauer

reaction (table 1.1). Each of the 01 biotype can be further subdivided into two major

serotypes, Ogawa and Inaba, based on variations in the polysaccharide component of the 0

antigen. Ogawa strains possesses only the A and B antigens while lnaba strains possesses

only the A and C antigens. A third and rare serotype, Hikojima possesses all three A, Band

C antigens (Kay et al., 1994).

2

The new serogroup 0139 Bengal was thought to have evolved as a result of

horizontal gene transfer between 01 and non-01 strains. The 0139 strain does not produce

01 lipopolysaccharide and lacks some of the genetic material necessary for production of the

01 antigen. It has potential for capsule expression and an increased capacity for spread and

proliferation within the environment (Morris and Cholera Laboratory Task Force, 1994).

Strains of V. cholerae identified by biochemical tests but do not agglutinate with 01

and 0139 antisera are referred to as non-01, non-0139 V. cholerae. The non-01, non-0139

strains can produce enterotoxins that are similar to cholera toxin (Morris, 1994). Although

this strain is not involved in cholera epidemics, it can be pathogenic and is frequently

associated with small sporadic outbreaks of diarrheal disease caused by the consumption of

contaminated shellfish and from a variety of extraintestinal infections (Morris, 1994).

Besides the somatic (0) antigen, all V. cholerae possess common heat-labile flagella

(H) antigen, and this property may be used to distinguish V. cholerae from all other vibrios

(Kay et al., 1994). Table 1.2 summarizes the different V. cholerae serogroups and their

antigenic determinants.

3

Table 1.1: Tests for differentiation of Classical and El Tor biotypes of V. cholerae serogroup 01

Hemolysis of sheep erythrocytes

Hemagglutination with fowl erythrocytes

Susceptibility to bacteriophage: i) classical phage (group IV) ii) El Tor phage (group V)

Sensitivity to Polymyxin B (50 U)

Produces acetoin in Voges Proskauer test (modified with 1% NaCl)

+

+

+ +

+

+

Table 1.2: V. cholerae serogroups and their antigenic determinants

Serogroup

Biotypes

Serotypes

01 Antigens

Toxin production

Polysaccharide capsul~

01

Classical El Tor

Ogawa Inaba

Hikojima

Ogawa- A and B Inaba- A and C

Hikojima- A, B and C

Produces cholera toxin

None

0139

None

None

Does not have 01 somatic

antigen

Produces cholera toxin

Yes

Non-01

None

Does not have 01 somatic

antigen

Usually does not produce cholera toxin, sometimes

produce other toxins

None

Adapted from Kay, B., Bopp, C. & Wells, J. (1994) In Vibrio cholerae and Cholera: Molecular to global perspectives (Eds, Wachsmuth, I., Blake, P. and Olsvik, 0.) American Society for Microbiology, Washington DC (table 1, pp. 4)

4

1.4 Ecology of V. cholerae

Until the late 1970s and early 1980s, it was believed that V. cholerae was highly

host-dependant and incapable of surviving longer than a few hours or days outside the

human intestine. However, studies have shown that V. cholerae is a normal inhabitant of

brackish water and estuarine systems (Colwell and Huq, 1994a). V. cholerae non-01, non-

0139 strains are frequently isolated from aquatic ecosystems while 01 and 0139 strains are

less frequently isolated. Outside of the host and in the aquatic environment, V. cholerae can

be found as free swimming cells, attached to aquatic plants, filamentous green algae,

copepods, crustaceans, insects and egg masses of chronomids. Environmental persistence of

V. cholerae within natural aquatic habitats during periods between epidemics is facilitated by

biofilm formation and entry into a viable but non-culturable (VBNC) state (Colwell and

Huq, 1994b ).

1.5 Viable but non-culturable (VBNC) state

V. cholerae 01 and non-01, non-0139 strains can enter into a VBNC state in

response to nutrient deprivation, elevated salinity and reduced temperatures. In this dormant

state, the V. cholerae cells are reduced in size, become ovoid and do not grow at all on

standard laboratory media used to isolate V. cholerae. However, Colwell eta/. (1994b) found

that when the VBNC cells of V. cholerae were inoculated into rabbit ileal loops or ingested

by human volunteers, it caused large accumulation of fluid in the rabbit ileal loops or

diarrhea in the human volunteers. This may explain the reason attempts to culture and isolate

V. cholerae from water sources between epidemics often fail. Since V. cholerae in the

VBNC state in environmental samples may not grow on conventional bacteriologic media, it

may go undetected unless other detection methods such as immunologic, molecular or direct I

microscopy are employed. Huq et al. (1990) showed that V. cholerae 01 could be detected

by direct immunofluorescence microscopy throughout the year in their water reservoirs even

when culturing could not isolate the organism itself. Rapid detection kits for V. cholerae that

do not require culture called CholeraScreen (a coagglutination test), Cholera DFA (direct

5

fluorescent monoclonal antibody staining kit) and Cholera SMART (a colorimetric

immunoassay) have been developed and these were also able to detect VBNC cells of

V. cholerae (Colwell and Huq, i994b).

1.6 Epidemiology of cholera

Cholera is the classic water-borne disease and an epidemiological study by John

Snow in 1849 showed the association of the disease with contaminated drinking water (Parsi,

200 I). Poor sewerage and sanitation practices that resulted in contamination of water

supplies used for drinking and food preparation are often the causes of cholera epidemics

(Mintz eta/., 1994). This mode of transmission is more common in less developed countries

under conditions of famine, war and natural disaster. Cholera transmission has also been

linked to drinking water from shallow wells, rivers, bottled water and ice (Parsi, 2001 ).

Food is another vehicle for transmission. Seafood, particularly raw or undercooked

shellfish harvested from sewage-contaminated seabeds, meat, cooked grains, fruit and

vegetables irrigated with raw sewerage have been implicated in cholera outbreaks (Parsi,

2001, Mintz eta/., 1994).

1. 7 Pathophysiology of cholera

Upon ingestion, V. cholerae cells colonize the small intestines, particularly the

duodenum and jejunum because the alkaline pH, nutrients and bile salts provide a suitable

environment. They adhere to the microvilli with the help of a long filamentous fimbriae

called toxin-coregulated pili (Tcp pili), hemagglutinins, mucinase and neuraminidase. Their

motility also enables them to penetrate the mucus layer. They penetrate the host cells by

elaborating a potent enterotoxin, called cholera toxin (CT), a 84 kDa protein consisting of 2

regions, )Nhich are the A region and B region (Sack eta/., 2004).

6

The B region (56 kDa) binds the glycolipid, GMl ganglioside on the host intestinal

epithelial cell membrane, enabling the A region (28 kDa) to penetrate the cell. The A region

will enzymatically transfer ADP-ribose from NAD to a GTP-binding regulatory protein

associated with membrane-bound adenylate cyclase enzyme. The ADP-ribosylation reaction

activates adenylate cyclase, which leads to an increase of intracellular cAMP levels. This is

followed by the secretion of H20, Na+, K+. cr and HC03- into the lumen (Kaper et al., 1994).

The patient may lose several litres of protein-free fluid and associated electrolytes,

bicarbonates and ions within hours. The watery diarrhea (rice-water stool) is speckled with

flakes of mucus, epithelial cells and contains large number of vibrios. Excessive loss of fluid

may lead to dehydration, uremia, metabolic acidosis, hypovolemic shock and subsequently,

death in untreated cases (Sack et al., 2004, Parsi, 2001). The severity of a cholera infection

depends on many factors, and it is increased with the absence of a local intestinal immunity,

a high infectious dose (> l 08 bacteria), low gastric acid production and possession of blood

group 0 (Sack et al., 2004, Parsi, 2001 ).

1.8 Clinical manifestations of cholera

Most individuals infected with V. cholerae have no symptoms or only mild diarrhea,

indistinguishable from other mild diarrheal diseases. Only a minority will develop cholera, a

disease characterized by the sudden onset of vomiting and profuse watery diarrhea,

following an incubation period of 6 to 48 hours. The most distinctive feature of cholera is the

painless purging of voluminous stools resembling rice-water and having a fishy odour. In

adults with severe cholera, the volume of watery stool may exceed 1 Llhour, leading to

severe dehydration, hypovolemic shock, metabolic acidosis and renal failure. This condition

may lead to death within a few hours of onset if rapid treatment is not given (Bennish, 1994,

Parsi, 2001 ).

7

1.9 Treatment of cholera

Without proper treatment the case-fatality for severe cholera is about 50% (Sack et

a!., 2004). Treatment is very simple and effective based on fluid replacement. Oral

rehydration solution (ORS) should be given for mild cases of diarrhea, while intravenous

fluid replacement using lactated Ringer's solution should be given for more severe cases

(Parsi, 2001 ). The use of antibiotics shortens the duration and severity of the illness. In most

cases, tetracycline and doxycycline is the antibiotic of choice. In cases of tetracycline

resistance, erythromycin, cotrimoxazole, ciprofloxacine, azithromycin and trimethoprim­

sulfamethoxazole may be used (Parsi, 2001 ).

1.10 Prevention of cholera

Transmission of V. cholerae through contaminated food and water can be prevented

by minimizing contamination and reducing survival and growth of the organism (Sack et a!.,

2004). The level of contamination can be minimized by practicing personal and domestic

hygiene such as hand washing with soap before food handling and safe sewage disposal.

Survival of the organism may be diminished by boiling or disinfecting drinking water and

proper cooking and refrigeration of food. Prophylactic prevention of cholera can be achieved

through the use of vaccines. Three oral vaccines (two inactivated and one live attenuated) are

currently available (Sack eta!., 2004). These vaccines have been licensed in some countries

and are mainly used by travellers. However, these vaccines are for V. cholerae serogroup 01

and do not give cross-protection against other serogroups such as 0139 or non-01, non-

0139. Although a number of vaccine candidates for V. cholerae serogroup 0139 are under

development (Ravichandran eta!., 2006), they are not commercially available. Prevention of

cholera using vaccines may be beneficial as a public health intervention if they are cost­

effective,' give long lasting protection and easy to administer.

8

1.11 Diagnosis of cholera

1.11.1 Conventional laboratory methods

Due to the clinical and epidemiological severity of cholera, rapid diagnosis of V.

cholerae in clinical samples, water and food is very critical so that appropriate monitoring

and effective preventive measures can be undertaken to prevent a cholera outbreak. The

conventional laboratory methods employed are culturing, microscopy and biochemical

testing.

Faecal specimens should be collected as early as possible in the illness, as the

number of V. cholerae begins to decline soon after onset of symptoms, and before initiation

of antibiotic therapy (Kay et al., 1994). Faecal specimens or rectal swabs should be collected

from patients into a sterile bottle containing transport medium such as alkaline peptone water

(APW). APW is also the standard medium for enrichment of V. cholerae as its high pH

condition preferentially supports the growth of vibrios while inhibiting other organisms and

it can be used to recover low levels ofvibrios in faecal specimens (Kay et al., 1994).

If specimen is collected on the first day of illness, the vibrios are likely to be present

in large numbers (107-109 CFU/ml), hence it is possible to make a provisional diagnosis by

direct microscopy examination of a film of faeces using dark-field or phase-contrast

microscopy (Kay et al., 1994). Vibrios are highly motile and can be seen darting about

rapidly. When specific antiserum is added to the film, the vibrios are immobilized.

For culture, fresh faecal specimens or APW-enriched specimens are plated onto

thiosulfate-citrate-bile salts-sucrose (TCBS) agar, a highly differential and selective medium

for V. cholerae. Its selective ingredients suppress the growth of most of the interfering

organisms such as coliforms, psedomonads, aeromonads and other Gram-positive bacteria

(WHO, 2002). The sucrose-fermenting V. cholerae grow on TCBS as large, slightly

flattened,1yellow, smooth colonies with a diameter of 2-4 mm.

9

Identification of 01 or 0139 serogroups is perfonned using slide agglutination test

with antiserum raised against V. cholerae 01 or 0139. Agglutination in polyvalent 01

antiserum provides presumptive identification of serogroup 01, which must then be

confirmed by agglutination with monovalent Ogawa and Inaba antisera. Identification of

serogroup 0139 should be perfonned with specific 0139 antiserum (Kay et al., 1994).

A series of biochemical tests may be used for further identification in addition to

serological identification. A crucial biochemical test for distinguishing V. cholerae from

other members of Enterobacteriaceae is the oxidase test. V. cholerae is oxidase-positive

while all Enterobacteriaceae are oxidase-negative. Other important traits that may be used to

distinguish V. cholerae include fermentation of glucose with acid production (without gas),

maltose, mannitol, sucrose and trehalose. In a string test, a loopful of V. cholerae suspended

in a drop of 0.5% aqueous deoxycholate gives a mucoid "string" when drawn. Inoculation of

V. cholerae into Kligler iron agar (KIA) or triple sugar iron agar (TSI) produces an alkaline

slant (K) over acid (A) butt, with no gas or H2S (Kay et al., 1994).

Conventional culture methods are time-consuming and labour-intensive. It takes at

least 2 days to obtain results and may lack sensitivity, especially for poorly handled samples

or clinical samples of patients previously treated with antibiotics as the number of viable and

culturable organisms may be diminished. Detection may also be difficult if the number of

viable organism is low, as in some environmental samples where V. cholerae can exist in a

metabolic state that is non-culturable, but still remain viable and capable of causing disease

(Colwell and Huq, 1994b).

10

1.11.2 Alternative methods of cholera diagnosis

1.11.2.1 Immunological-based tests for cholera

As an alternative to conventional culture methods, several rapid immunological­

based tests have been developed. The earlier studies employed antibodies against V cholerae

01 antigen in enzyme-linked immunosorbent assay (ELISA) format (Martinez-Govea et al.,

2001, Osek eta/., 1992, Bhadra eta/., 1991, Gustafsson, 1984, Cooper eta/., 1983), bead­

ELISA format (Ramamurthy eta/., 1996, Ramamurthy eta/., 1992, Uesaka et al., 1992,

Cooper et a/., 1983), immunofluorescence microscopy (Goel et a/., 2005, Hasan et a/.,

1994a, Huq eta/., 1990), coagglutination test (Goel et al., 2005, Hasan eta/., 1995, Colwell

eta/., 1992, Rahman eta/., 1989, Rahman eta/., 1987) and colloidal gold-based colorimetric

immunoassay (Hasan et a/., 1994b). Immunoassays for V cholerae 0139 have also been

developed using coagglutination test (Qadri et al., 1994), immunofluorescence microscopy

(Hasan et al., 1995), colloidal gold-based colorimetric test (Qadri eta/., 1995) and dot-blot

ELISA (Chaicumpa eta/., 1998).

More recently, antibody-based biosensors, called immunosensors, have been

described. Jyoung et a/. (2006) immobilized monoclonal antibodies against V cholerae 01

on a self-assembled monolayer (SAM), whereby binding of 01 antigen to the antibody can

be detected using surface plasmon resonance (SPR) spectroscopy. Rao eta/. (2006) utilized

an indirect ELISA method in developing an amperometric immunosensor for detection of V

cholerae 01 on disposable screen-printed electrodes.

Most of the immunological assays offer the advantages of speed, simpler reagents

and equipments, better sensitivity and specificity over culture methods. Several tests such as

CholeraScreen, Cholera DF A and Cholera SMART have been reported to be able to detect

VBNC cells from faecal specimens (Colwell and Huq, 1994b). However, only a few tests are

commerdally available, namely SMART™ and Medicos™ Cholera Dipstick to detect the

01 serogroup but these tests are quite expensive and well beyond the reach of medical

providers in areas at risk of cholera (Kalluri et al., 2006).

11

1.11.2.2 Molecular-based tests for cholera

Recently, there has been a move to adopt molecular-based methods as a gold

standard to replace the traditional culture methods. The use of molecular techniques that rely

on nucleic acid amplification, such as polymerase chain reaction (PCR), ligase chain reaction

(LCR), transcription-mediated amplification (TMA), strand displacement amplification

(SDA) and nucleic acid sequence-based amplification (NASBA) have greatly improved the

speed, sensitivity and specificity of diagnostic tests. This, in turn, would facilitate early and

informed decision-making related to patient management, infection control, treatment and

prevention.

PCR assays have been developed to detect the cholera enterotoxin operon, since

most of the cholera pandemic and epidemics were associated with enterotoxin producing

V. cholerae (Koch et al., 1993, Fields et al., 1992, Shirai et a/., 1991). Subsequently, a

multiplex PCR assay was developed to detect toxigenic strains of V. cholerae based on the

cholera toxin gene and to identify its biotype based on the hyl gene (Shangkuan et al., 1995).

As the new cholera pandemic involving the serogroup 0139 began to emerge,

various PCR tests were developed to detect this new serogroup 0139 alone (Albert et a/.,

1997), or both the serogroups 0139 and 01 (Gubala, 2006, Lipp et al., 2003, Lyon, 2001,

Singh et al., 2001, Albert et al., 1997, Rivera eta!., 1995). Besides detecting the serogroups

01 and 0139, Nandi eta!. (2000) and DiPinto eta!. (2005) have developed PCR assays

based on non-virulence genes such as the ompW and collagenase gene, respectively, which

were able to detect certain non-toxigenic V. cholerae strains of the serogroups non-0 1, non-

0139. This serogroup is often found in environmental samples and causes sporadic and

localized diarrheal outbreaks (Vital-Brazil eta!., 2002, Kaper eta!., 1994).

12

11.2.2.1 Characteristics and advantages of molecular-based tests over conventional

methods

The advantage of PCR over conventional culturing in detecting viable but non-

ulturable (VBNC) cells of V. cholerae was proven in a study by Chaiyanan et al. (200 l ).

~onservation of cholera toxin-associated genes like ctxA, tepA, toxR and zot in chromosomal

)NA of VBNC cells was demonstrated using PCR. It was shown that VBNC cells retained

heir viability, along with their genes and chromosomal integrity up to one year.

PCR assays are rapid, highly sensitive, specific and can detect more than one gene at

the same time. Although the advantages ofPCR-based test over conventional culture method

are numerous, there are certain limitations to the test. PCR requires the use of a thermal

cycler and even with recent advances in this field, the cost and size of a thermal cycler limit

its use in laboratories with limited resources (Lai et a!., 2006). In addition, PCR reagents

must be transported and stored at -20°C and maintaining a constant cold chain may not be

possible when transporting to far or remote places.

These are important considerations in developing a diagnostic test for cholera, as

cholera is a disease that affects most of the poor under-developed countries, or countries

ravaged by war or natural disasters. Hence, there are not many PCR-based tests for cholera

in the market, while those that are available commercially are immunochromatographic-

based assays, which can be quite expensive and still require refrigeration for long term

storage (Kalluri et al., 2006).

PCR can amplify DNA from both live (viable) and dead (non-viable) cells, since

DNA can remain intact and undegraded long after cell death. Thus, PCR-based tests cannot

differentiate viable from non-viable cells. Since cholera is transmitted mainly by

contaminated food and water, detection of viable cells from these samples is desirable during I

an epidemic to identify the source of infection and implement control measures. It is

important that positive test results are associated with live cells and not the remains of dead

cell. Identification of live cells would also help clinicians in monitoring the effectiveness of

l3

antimicrobial treatments in patients (Bej et al., 1996). Detection of viable microorganisms

after sterilization procedures is also important in the medical and food industry to ensure

effectiveness of the process.

RNA has been suggested as a suitable indicator of cell viability as it rapidly degrades

after cell death (Sheridan et al., 1999, Sheridan et al., 1998). Detection of RNA is possible

through reverse-transcription PCR (RT-PCR), a modification of the PCR process, which first

converts the RNA to eDNA, and subsequently the eDNA is amplified by PCR. RT-PCR is a

multiple step process and even small amounts of DNA in the RNA sample might get

amplified, thus giving a false-positive result. Therefore, all RNA samples need to be treated

with DNase enzyme prior to RT-PCR.

1.11.2.3 Nucleic acid sequence-based amplification (NASBA)

Besides RT-PCR, another RNA-based amplification technique is nucleic acid

sequence-based amplification (NASBA). NASBA is an isothermal amplification assay that

specifically amplifies RNA. The reaction occurs at a relatively low temperature of 41 oc that

does not promote the denaturation of double-stranded DNA, hence DNA is not amplified

(Deiman et al., 2002). Furthermore, it can be performed using a simple water bath or heating

block. NASBA produces more amplicons in a shorter time than PCR, requiring only 60- 90

minutes of assay time, compared to 2- 3 hours for RT-PCR. Amplification of approximately

106 to 109-fold is obtained within 90 minutes (Compton, 1991).

The single-stranded RNA amplicons can be detected using various methods, such as

agarose gel electrophoresis (Jean et al., 2004, Jean et al., 2002b), molecular beacon-based

real-time amplification technique (Loens et al., 2008, Churruca et al., 2007, Deiman et al.,

2007, Fykse et al., 2007, Nadal et al., 2007) and probe hybridisation techniques, such as

Northern blotting (Jean et al., 2004, Jean et al., 2002b), ELISA (Lau et al., 2008, Jean et al.,

2002a, Jean et al., 2002b), electrochemiluminescence (ECL) (Lau et al., 2008, Collins et al.,

2003, Hibbitts et al., 2003), enzyme-linked gel assay (ELGA) (Lau et al., 2008, Hibbitts et

14

al., 2003) and biosensors (Baeumner et al., 2004a, Baeumner et al., 2004b, Zaytseva et al.,

2004). These features make NASBA a suitable molecular-based test for cholera. Figure 1.1

shows an overview of the NASBA reaction.

NASBA was first reviewed in 1991 by Compton (1991), who described the salient

features of the technology and its applications. Kievits et al. (1991) was among the earliest to

describe the use ofNASBA for the diagnosis ofHIV-1 infection. Since NASBA is an RNA

amplification technology, it is suitable for detection of RNA viruses, such as norovirus (Kou

et al., 2006, Patterson et al., 2006, Rutjes et al., 2006), enterovirus (Casper et al., 2005,

Landry et al., 2005), hepatitis A virus (Abd el-Galil et al., 2005) and cytomegalovirus

(Bergallo et al., 2006, Revello et al., 2003).

NASBA provided researchers with a way of detecting viable microorganisms in

clinical samples, food and environmental samples, as presence of intact RNA is a valuable

indicator of viability. Detection of viable microorganisms is important for screening of

contaminated food (Cook, 2003, Gore et al., 2003) and environmental samples (Fykse et al.,

2007, Rutjes et al., 2006, Baeumner et al., 2003); monitoring the clinical course of a disease

(Yoo et al., 2005); and assessing the efficacy of antimicrobials and other treatments (de

Vries et al., 2006).

A real-time NASBA assay for V. cholerae was developed by Fykse et a!. (2007)

targeting genes encoding cholera (etxA), toxin-corregulated pilus (tepA), ctxA toxin regulator

(toxR), hemolysin (hlyA) and the 60 kDa chaperonin product (groEL). The assay was able to

detect V. eholerae from various environmental water samples and has a strong potential for

detecting toxigenic strains using the tepA and etxA markers.

Besides detection of medically important microorganisms, other clinical applications

of NASBA include detection of biomarkers and important human genes. NASBA has also

been used to detect disease-causing microorganisms in the marine, agriculture and livestock

industries (Lau et al., 2008, Lau et al., 2006, Olmos et al., 2007, Teng et al., 2006).

15

RNA(+)

T7 primer '-

1

NON-CYCLIC PHASE

t Reverse transcriptase

TIRNA polymerase

------...

'-

1 Reverse T transcriptase

~ RnaseH

eDNA

1 Reverse • tra.nscriptase

I TIRNA T polymerase

....................... ············-········ ••••w•••••••••••••••• .....•............... .....................

RNA(-)

CYCLIC PHASE

Figure 1.1: Overview of the NASBA reaction

16

1 Reverse + transcriptase

1.12 Rationale of the study

Most of the molecular-based diagnostic tests that have been developed for

V. cholerae are either PCR or real-time based PCR assays. These assays require constant

cold chain for transport and storage of the PCR reagents, skilled laboratory technicians and

sophisticated equipments to perform the tests. Moreover PCR assays detect DNA, which

may still be present in dead cells; therefore it cannot be used to differentiate between viable

and non-viable cells. Identification of viable V. cholerae is important especially during a

cholera epidemic, where detection of viable cells from food and water samples is desirable to

identify the source of infection and implement control measures. It is also useful for testing

environmental samples for reservoirs of viable V. cholerae.

We have identified a non-virulence gene called lolB (hemM), which codes for an

outer membrane protein in V. cholerae. The lolB gene sequence has been submitted to the

Genbank under the accession number AF227752. The lolB gene sequence was found to be

highly conserved among the different serogroups of V. cholerae. A PCR assay was

developed that was able to detect 01, 0139 and non-01, non-0139 serogroups of

V. cholerae from clinical and environmental samples with 98.5% sensitivity and 100%

specificity (Lalitha et al., 2008).

The present study used the lolB gene as a candidate in the development of a cholera

NASBA-ELISA assay for detection of V. cholerae. The cholera NASBA-ELISA assay with

biosensor detection would be a suitable detection method for viable V. cholerae as it does

not require expensive and specialized equipments. The present study also aims to make

NASBA easy to perform and to eliminate the need for cold storage of NASBA reagents

through the use of our patent-pending thermostabilization technology (Ravichandran et al.,

2005), which has been successfully applied in thermostabilization PCR assays for typhoid

and cholera (Aziah et al., 2007, Ravichandran et al., 2003). To the best of our knowledge,

this is the first time a them1ostabilized cholera NASBA-ELISA assay featuring the use of a

portable biosensor is described.

17

1.13 Objectives ofthe study

Phase 1: Development and optimization of a cholera NASBA assay

1. To design specific primers and probes for cholera NASBA assay

2. To construct in vitro RNA transcripts as positive control

3. To develop and optimize a cholera NASBA assay targeting the lolB gene of V. cholerae

4. To analyze the NASBA reaction and its amplicons

5. To test the suitability of lolB mRNA as an indicator of cell viability

Phase 2: Development and optimization of ELISA for detection of cholera NASBA

amplicons (cholera NASBA-ELISA)

6. To develop and to optimize an ELISA test for detection of NASBA amplicons by

comparison of two ELISA formats

7. To evaluate the cholera NASBA-ELISA assay with spiked stool samples

8. To calculate the diagnostic sensitivity, specificity, positive predictive value (PPV) and

negative predictive value (NPV) of the clinical evaluation

Phase 3: Development of an electrochemical biosensor detection for cholera NASBA

ELISA assay

9. To optimize detection of the NASBA-ELISA signal with an electrochemical biosensor

10. To compare the biosensor results with spectrophotometric measurements

Phase 4: Optimization of a thermostabilized NASBA mix

10. To optimize the enzymes and enzyme stabilizer concentrations for thermostabilization of

the NA5BA mix

11. To detennine the stability of the thermostabilized NASBA m1x stored at different

temperatures over a certain period of time

18

1.14 Overview of the study

Development and optimization of a NASBA assay targeting the

lo!B gene of Vibrio cholerae

• Detection ofNASBA amplicons by

agarose gel electrophoresis

• Determination of

analytical specificity

• •

Determination of analytical sensitivity

Analysis ofNASBA reaction and amplicons

••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••

Development and optimization of ELISA for detection ofNASBA amplicons (NASBA-ELISA)

• Determination of analytical specificity and sensitivity

• Evaluation ofNASBA-ELISA with spiked stool samples

• Determination of diagnostic sensitivity, specificity,

PPV, NPV and limit of detection (LOD)

••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••

Optimization of an electrochemical biosensor method for detection of ELISA reaction

• Comparison of biosensor and

spectrophotometry results

• Determination of analytical sensitivity and specificity

••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••

Optimization of a thermostabilized formulation for lyophilization of the NASBA mix

• Periodic stability evaluation of the

lyophilized NASBA mix

19

• Determination of

analytical sensitivity

2.1 Materials

2.1.1 Bacterial strains

2.1.1.1 Reference strains

CHAPTER2

MATERIALS AND METHODS

Six V. cholerae strains comprising of 01, 0139 and non-01, non-0139 serogroups

were used as reference strains for optimization and evaluation of the sensitivity of the

NASBA assay. Six other Vibrio species strains and 29 enteric pathogen strains were used as

reference strains for evaluating the specificity of the NASBA assay. The V. cholerae, Vibrio

spp. and 10 enteric pathogen strains were obtained from Prof. Mitsuaki Nishibuchi, Centre

for Southeast Asia Studies, Kyoto University, Japan as a gift. The remaining 19 enteric

pathogen strains were obtained from the culture collection of the Department of

Microbiology and Parasitology, Universiti Sains Malaysia, Malaysia. Details of the reference

strains used in this study are given in table 2.1.

2.1.1.2 Clinical isolates

For evaluation of the NASBA assay using spiked stool samples, 110 well­

characterized strains of V. cholerae isolated from clinical samples were obtained from Centre

for Southeast Asia Studies, Kyoto University, Japan; Communicable Diseases Hospital,

Thandiarpettai, Chennai, India; Hospital Universiti Sains Malaysia (HUSM) and General

Hospital Kota Bharu (GHKB), Kota Bharu, Kelantan; National Public Health Laboratory

(MKAK), Ministry of Health, Sungai Buloh; and Institute for Medical Research (IMR),

Selangor, Malaysia.

In addition, 45 well-characterized strains of other Vibrio species and various enteric

pathogens isolated from clinical samples were also obtained from Centre for Southeast Asia

20

Studies, Kyoto University, Japan; Hospital Universiti Sains Malaysia (HUSM), Kota Bharu,

Kelantan and Institute of Medical Research (IMR), Selangor, Malaysia.

All 41 reference strains were also included in the evaluation of the NASBA assay

using spiked stool samples as these were originally isolated from clinical samples. Details of

the clinical isolates used in the study are given in table 2.2.

2.1.1.3 Growth and maintenance of bacterial strains

All bacterial strains were preserved and maintained as glycerol stock in LB broth

with 15% glycerol and kept at -70°C. For experimental purposes, the bacterial strains were

revived from glycerol stocks by inoculating a loopful of culture into LB broth and incubated

overnight at 37°C. For V. cholerae strains, the overnight cultures were then subcultured onto

TCBS agar while for other enteric bacteria, the overnight cultures were subcultured onto LB

agar and incubated overnight at 37°C. V. cholerae appeared as yellow, convexed and mucoid

colonies on TCBS agar. A single colony was picked with a sterile wire loop, streaked onto

LB agar and incubated overnight at 3 7°C again. These cultures were used to perform the

NASBA assay.

2.1.2 Culture Media

Culture media were prepared using molecular biology or biotechnology grade

chemicals. Distilled water was used for preparing media solutions. The appropriate media

were autoclaved at 121 °C, 15 psi for 15 minutes. Sterility check was performed by

incubating the media at 37°C overnight. Sterile agar media were stored at 4°C while broth

and other liquid media were stored at room temperature. The list of culture media used in

this study is given in appendix A.

21

Table 2.1: Details of the reference strains used in this study

Vibrio Centre for V. cholerae, 01 Classical 2 cholerae Southeast Asia V. cholerae, 01 El Tor 2 (n =6) Studies, Kyoto V. cholerae, 0139 Bengal 1

University, Japan V. cholerae, non-01, non-0139 1

Other Centre for V. cincinnatiensis 1 Vibrio spp. Southeast Asia V. fluvial is 1 (n= 6) Studies, Kyoto V. furnissii 1

University, Japan V. mimicus 1 V. parahaemolyticus 1 V. vulnificus 1

Enteric Centre for Aeromonas hydrophila 1 pathogens Southeast Asia Escherichia coli 2 (n = 29) Studies, Kyoto Pleisiomonas shigelloides 1

University, Japan Salmonella enteritidis 1 Salmonella typhi 1 Shigella boydii 1 Shigella dysenteriae 1 Shigella jlexneri 1 Shigella sonnei 1

Department of Acinetobacter spp. 1 Microbiology Acinetobacter baumanii 1 and Parasitology, Bacillus cereus 1 Universiti Sains Bacillus subtilis 1 Malaysia Burkholderia pseudo mallei 1

Citrobacter freundii 1 Enterobacter cloacae 1 Klebsiella spp. 1 Klebsiella pneumoniae 1 Morganella morganii 1 Proteus mirabilis 1 Proteus vulgaris 1 Providencia stuartii 1 Pseudomonas aeruginosa 1 Salmonella spp. 1 Salmonella para typhi A 1 Salmonella para typhi B 1 Serratia marsescens 1 Yersinia enterocolitica 1

22

Table 2.2: Details of the clinical isolates used in this study

Vibrio Hospital Universiti Sains V cholerae, 01 8 cholerae Malaysia (HUSM), (n = 116) Kelantan, Malaysia

General Hospital, Kota V cholerae, 01 17 Bharu (GHKB), Kelantan, Malaysia

National Public Health V. cholerae, 01 6 Laboratory, Ministry of Health, Sungai Buloh, Selangor, Malaysia

Institute for Medical V cholerae, 01 55 Research (IMR), V cholerae, 0139 Bengal 1 Selangor, Malaysia V cholerae, non-01, non-0139 3

Communicable Diseases V cholerae, 0139 Bengal 9 Hospital, Thandiarpettai, Chennai, India

Centre for Southeast V cholerae, 01 14 Asia Studies, Kyoto V cholerae, 0139 Bengal 2 University, Japan V. cholerae, non-01, non-0139 1

Other Centre for Southeast V. cincinnatiensis 1 Vibrio Asia Studies, Kyoto V. jluvialis 3 spp. University, Japan V. furnissii 2 (n =12) V. mimicus 2

V parahaemolyticus 1 V. vulnificus 2

Institute for Medical V vulnificus Research (IMR), Selangor, Malaysia

Enteric Centre for Southeast Aeromonas hydrophila 1 pathogens Asia Studies, Kyoto Escherichia coli 4 (n = 68) University, Japan Pleisiomonas shigelloides 1

Salmonella enteritidis 1 Salmonella typhi 1 Shigella boydii 1 Shigella dysenteriae 1 Shigella jlexneri 1 Shigella sonnei 1

23

Table 2.2 (continued)

Enteric Department of Acinetobacter spp. 1 pathogens Microbiology and Acinetobacter baumannii 1 (n = 68) Parasitology, Universiti Bacillus cereus 1

Sains Malaysia Bacillus subtilis 1 Burkholderia pseudomallei 1 Citrobacter freundii 2 Enterobacter cloacae 1 Escherichia coli 1 Klebsiella spp. 1 Klebsiella pneumoniae 1 Morganella morganii 1 Proteus vulgaris 1 Pseudomonas aeruginosa 2 Salmonella enteritidis 1 Salmonella typhi 1 Serratia marsescens 1 Shigella boydii 1 Staphylococcus aureus 1 Streptococcus group G 1

Institute for Medical Aeromonas hydrophila 1 Research (IMR), Acenitobacter baumanii 1 Selangor, Malaysia Bacillus cereus 1

Burkholderia pseudomallei 1 Enterococus faecalis 1 Klebsiella spp. 1 Klebsiella pneumoniae 1 Proteus mirabilis 1 Providencia stuartii 1 Salmonella spp. 4 Salmonella enteritidis 1 Salmonella para typhi A 5 Salmonella para typhi B 3 Salmonella typhi 4 Shigella boydii 2 Shigella dysenteriae 2 Shigella jlexneri 1 Shigella sonnei 2 Staphylococcus aureus 1 Yersinia enterocolitica 1

24