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LEPTOSPIRAL PROTEINS INDUCED IN-VIVO AND ITS APPLICATION IN THE DEVELOPMENT
OF ANTIBODY AND ANTIGEN DETECTION TESTS FOR ACUTE LEPTOSPIROSIS
CHANG CHIAT HAN
UNIVERSITI SAINS MALAYSIA
2018
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LEPTOSPIRAL PROTEINS INDUCED IN-VIVO AND ITS APPLICATION IN THE DEVELOPMENT
OF ANTIBODY AND ANTIGEN DETECTION TESTS FOR ACUTE LEPTOSPIROSIS
by
CHANG CHIAT HAN
Thesis submitted in fulfillment of the requirements for the degree of
Doctor of Philosophy
May 2018
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ACKNOWLEDGEMENT
I would like express my highest gratitude to Prof. Dr. Rahmah Noordin, the supervisor of my Ph.D study for her continuous inspiration and enthusiasm towards this study.
Her generous sharing of her rich knowledge, experience and the passion to transform the knowledge for the benefit of bottom million people absolutely inspires me and would certainly be the compass in my future life. Also, it is my honor to be co- supervised by Dr. Mehdi Riazi. His encouragement and positive energy are the force for me to keep exploring in research field of leptospirosis.
I appreciate the generous support from laboratory staffs of INFORMM, particularly En. Mohd Hafiznur, Pn. Sabariah, Pn. Dyana, Pn. Ezzati and Pn. Azimah, as well as administrative staffs, especially Pn. Faizulkisnu, Pn. Nurul' Jannah, En. Adli, En. Hafriz and Pn. Siti Zainaf that make this study smooth throughout the period.
Nonetheless, it is grateful to have lab- and soul mates from Proteomic Labs, including Dr. Syahida, Dr. Syazwan, Dr. Teh (Ai Ying), Dr. Akbar, Dr. Atefeh, Dr. Monsur, Dr.
Nurulhasanah, Naqiuyah, Kang Zi, Sam, Sam (Xin Hui) and Dinesh to be with me during my ups and downs. The success and pride of this study is dedicated to my parents and sibling for their great support, patience and encouragement throughout this journey. It is their faith on me that make this study happened and accomplishes by now.
The present study was funded by Science Fund (no. 305/CIPPM/613611) from the Ministry of Science, Technology and Innovation, and Prototype Research Grant Scheme (203/CIPPM/6740024) under Flood Disaster Research Program of Ministry of Higher Education to Prof. Dr. Rahmah Noordin. Also, I am very thankful to the financial support from MyPhD scholarship, Ministry of Education Malaysia.
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TABLE OF CONTENTS
ACKNOWLEDGEMENT ... ii
LIST OF TABLES ... xv
LIST OF FIGURES ... xviii
LIST OF ABBREVIATIONS ... xxii
ABSTRAK ... xxiv
ABSTRACT ... xxvi
CHAPTER ONE: INTRODUCTION ... 1
CHAPTER TWO: LITERATURE REVIEW ... 4
2.1 World Epidemiology ... 4
2.1.1 Leptospirosis in Malaysia ... 6
2.1.2 Outbreaks and Case Reports in Malaysia... 9
2.2 Classification and Typing of Leptospira ... 11
2.2.1 Taxonomy ... 11
2.2.2 Classification of Leptospira Species And Subspecies ... 12
2.3 Animal Reservoir for Pathogenic Leptospira spp. ... 15
2.4 Anatomy of Leptospira ... 17
2.4.1 Lipopolysaccharide ... 17
2.4.2 Outer Membrane Proteins ... 21
2.4.3 Periplasm ... 22
2.4.4 Endoflagellum ... 23
2.5 Pathogenesis ... 23
2.5.1 Invasion ... 25
2.5.2 Dissemination ... 26
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2.5.3 Blood Vessel Transpass ... 26
2.5.4 Organ Colonization ... 27
2.5.5 Virulence Mechanism ... 28
2.6 Symptom and Clinical Manifestation ... 29
2.6.1 Acute Phase (Leptospiraemic Phase) ... 29
2.6.2 Convalescent Phase (Immune Phase)... 30
2.7 Diagnosis ... 33
2.7.1 Clinical Diagnosis ... 33
2.7.2 Laboratory Diagnosis ... 36
2.7.2(a) Type of Sample and Time of Sampling ... 36
2.7.2(b) Direct Visualization ... 39
2.7.2(c) Culture ... 40
2.7.2(d) Microscopic Agglutination Test ... 43
2.7.2(e) Gene Amplification ... 45
2.7.2(f) Immunoassays ... 47
2.8 Treatment and Prevention ... 51
2.9 Statement of The Problems And Rationale of The Study ... 52
2.10 Objectives of the Study ... 60
CHAPTER THREE: MATERIALS AND METHODS ... 61
3.1 Study Design ... 61
3.1.1 Phase I ... 61
3.1.2 Phase II ... 61
3.1.3 Phase III ... 64
3.1.4 Phase IV ... 65
3.1.5 Phase V ... 66
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3.2 Materials ... 67
3.2.1 Microorganism Strains ... 67
3.2.2 Plasmids and Oligonucleotides ... 67
3.2.3 Kits ... 67
3.2.4 Computer Softwares ... 67
3.2.5 Reagents for Serum Preabsorption ... 74
3.2.5(a) Maltose ... 74
3.2.5(b) Magnesium Sulfate ... 74
3.2.5(c) LB/MgSO4/Maltose Broth ... 74
3.2.5(d) Washing Solution ... 74
3.2.5(e) Blocking Solution ... 75
3.2.6 Reagents for Enzyme-Linked Immunosorbent Assay... 75
3.2.6(a) Carbonate/bicarbonate Coating Buffer ... 75
3.2.6(b) Blocking Solution ... 75
3.2.6(c) Substrate Solution for ELISA ... 75
3.2.7 Reagents for Immunoscreening of Leptospira gDNA Expression Library ... 76
3.2.7(a) LB/MgSO4 Agar ... 76
3.2.7(b) LB/MgSO4 Soft Top Agarose... 76
3.2.7(c) Lambda Dilution Buffer ... 76
3.2.7(d) Blocking Solution ... 77
3.2.7(e) Enhanced Chemiluminescence Substrate ... 77
3.2.8 Reagents for Conversion of Lambda TriplEx to Plasmid TriplEx ... 77
3.2.8(a) LB/Ampicillin Agar Plate ... 77
3.2.8(b) Magnesium Chloride Solution ... 78
3.2.9 Reagents for Native Purification of Recombinant Proteins ... 78
3.2.9(a) DNAse I Solution ... 78
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3.2.9(b) Lysozyme... 79
3.2.9(c) Buffers for Native Purification ... 79
3.2.10 Reagents for Denaturing Purification of Recombinant Leptospiral Protein Antigens ... 81
3.2.11 Reagents for Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis ... 81
3.2.11(a) Resolving Buffer... 81
3.2.11(b) Stacking Buffer ... 81
3.2.11(c) Running Buffer ... 82
3.2.11(d) Ammonium Persulfate ... 82
3.2.11(e) Sample Buffer ... 82
3.2.11(f) Coomassie Brilliant Blue Staining Solution ... 83
3.2.11(g) Destaining Solution ... 83
3.2.12 Reagents for Western Blot ... 83
3.2.12(a) Towbin Transfer Buffer ... 83
3.2.12(b) Blocking Solution for Western Blot ... 83
3.2.12(c) Developer and Fixer ... 84
3.2.13 Reagents for OFFGEL Fractionator ... 84
3.2.13(a) Protein OFFGEL Stock Solution (1.25x) ... 84
3.2.13(b) Protein IPG Strip Rehydration Solution ... 85
3.2.13(c) Bromophenol Blue (0.2%) ... 85
3.2.14 Reagents for Lateral Flow Dipstick Test ... 85
3.2.14(a) Blocking Solution ... 85
3.2.14(b) Chase Buffer ... 85
3.2.15 Reagent for Refolding of Denatured Protein ... 86
3.2.15(a) PBS-Arginine... 86
3.2.16 Reagents for Rabbit Polyclonal Antibodies Production ... 86
3.2.16(a) Freund’s Adjuvant ... 86
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3.2.17 Reagents for Affinity Purification of Target-Specific
Antibody ... 86
3.2.17(a) Tris Buffered Saline for Affinity Purification ... 86
3.2.17(b) Phosphate Buffered Saline for Affinity Purification ... 87
3.2.17(c) Glycine-HCl Elution Buffer ... 87
3.3 Methodology ... 88
3.3.1 Evaluation of Two Commercial Leptospirosis Diagnostic Kits Commonly Used in Malaysia and Categorization of Serum Panel ... 88
3.3.1(a) Sample Collection... 88
3.3.1(b) VISITECT-LEPTO ... 92
3.3.1(c) Leptorapide ... 92
3.3.1(d) Panbio Dengue IgM Capture ELISA ... 94
3.3.1(e) Serodia-TPPA ... 95
3.3.1(f) Data Analysis ... 96
3.3.2 Immunoscreening for Leptospiral In Vivo Induced Antigen... 96
3.3.2(a) In Vitro Antigen Preparation ... 96
3.3.2(a)(i) Determination of Protein Concentration by Bio-Rad RC DCTM Protein Assay ... 98
3.3.2(a)(ii) Coating of Antigen on Polystyrene Microsphere Beads ... 99
3.3.2(b) Serum Selection and Absorption ... 100
3.3.2(c) Indirect ELISA ... 101
3.3.2(d) Construction of Leptospira Genomic DNA Expression Library ... 102
3.3.2(e) Immunoscreening of Leptospira Genomic Expression Library ... 103
3.3.2(f) In Vivo Excision and Sequence Determination of Recombinant Phage Clones ... 105
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3.3.2(f)(i) Extraction of Recombinant Plasmid from Escherichia coli and
DNA Sequencing ... 107
3.3.2(f)(ii) Determination of Plasmid DNA Concentration ... 108
3.3.3 Bioinformatic Analysis of Leptospiral In Vivo Induced Antigens ... 109
3.3.3(a) Nucleotide and Protein Basic Local Alignment Search Tool ... 109
3.3.3(b) Multiple Sequence Alignment of DNA and Proteins ... 109
3.3.3(c) Phylogenetic Analysis ... 110
3.3.3(d) Prediction of Antibody Epitopes ... 112
3.3.4 Expression and Purification of Leptospiral Protein Antigens ... 112
3.3.4(a) Preparation of E. coli BL21 (DE3) Competent Cells ... 112
3.3.4(b) Transformation of Plasmid DNA into E. coli BL21 (DE3) Expression Host by Heat Shock Method ... 113
3.3.4(c) Long Term Storage of E. coli Recombinant Clone ... 113
3.3.4(d) Expression of Recombinant Leptospiral Protein Antigens ... 114
3.3.4(e) Native Purification of Recombinant Leptospiral Protein Antigens ... 114
3.3.4(f) Denaturing Purification of Recombinant Leptospiral Protein Antigens ... 115
3.3.4(g) Refolding of Denatured Recombinant LepS8A1FL Protein ... 116
3.3.4(h) Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis ... 116
3.3.4(i) Pooling and Buffer Exchange of Purified Recombinant Leptospiral Protein Antigens ... 118
3.3.4(j) Western Blot ... 119
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3.3.4(k) Confirmation of Recombinant LepS8A1FL by
MALDI-TOF/TOF Analysis... 120
3.3.4(l) Isoelectric Point Determination of Recombinant LepS8A1FL and LepS8A134 ... 120
3.3.5 Evaluation of Diagnostic Performance of Recombinant LepS8A1FL and other Leptospira Protein Antigens ... 121
3.3.5(a) IgM Western Blot ... 121
3.3.5(b) IgM Enzyme-Linked Immunosorbent Assay ... 122
3.3.5(c) Data Analysis ... 123
3.3.6 Development of A Rapid Antibody Detection Test in IgM Lateral Flow Dipstick Format for Acute Leptospirosis Using Recombinant LepS8A1FL Protein Antigen ... 124
3.3.6(a) Configuration of Antibody Detection Lateral Flow Dipstick ... 124
3.3.6(b) Test Procedure for Lateral Flow Dipstick Antibody Detection Assay ... 124
3.3.6(c) Data Analysis ... 125
3.3.7 Development of Rapid Antigen Detection Tests for Acute Leptospirosis in Lateral Flow Dipstick Format ... 126
3.3.7(a) Production of Rabbit Polyclonal Antibodies Against Leptospiral Protein Antigens ... 126
3.3.7(a)(i) Preparation of Immunogens ... 126
3.3.7(a)(ii) Electro-Elution of Protein Immunogens from Gel Slices ... 127
3.3.7(a)(iii) Rabbit Immunization ... 128
3.3.7(a)(iv) Immunoreactivity of Rabbit Antisera Towards Their Respective Leptospira Antigen Proteins by Western Blot... 129
3.3.7(a)(v) Titer Determination of Anti- Leptospiral Antibodies ... 130
3.3.7(b) Immunodetection of Endogenous LepS8A1FL Protein in Leptospira Lysate ... 131
3.3.7(b)(i) Affinity Purification of LepS8A1FL-specific Antibody ... 131
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3.3.7(b)(ii) Indirect Demonstration of LepS8A1FL as An In Vivo Induced Antigen in Pathogenic Leptospira
spp. ... 132 3.3.7(c) Gold Nanoparticle Conjugation to Rabbit
Immunoglobulin G ... 133 3.3.7(c)(i) Purification of Immunoglobulin G
Fraction from Rabbit Antiserum ... 133 3.3.7(c)(ii) Production of Gold Nanoparticle-
Conjugated Rabbit
Immunoglobulin G ... 134 3.3.7(d) Development of A Rapid Serological Antigen
Detection Test for Diagnosis of Acute
Leptospirosis ... 134 3.3.7(d)(i) Configuration of Antigen
Detection Lateral Flow Dipstick
Assay ... 134 3.3.7(d)(ii) Sample Preparation ... 135 3.3.7(d)(iii) Test Procedure for Antigen
Detection Lateral Flow Dipstick
Assay ... 137 3.3.7(d)(iv) Data Analysis ... 138
CHAPTER FOUR: RESULTS ... 139 4.1 Establishment of Serum Panel and Evaluation of Commercial Rapid
Diagnostic Kits for Acute Leptospirosis ... 139 4.2 Investigation of Novel Diagnostic Markers for Acute Leptospirosis
Using In Vivo Induced Antigen Technology ... 145 4.2.1 Serum Preabsorption ... 145 4.2.2 Immunoscreening of Leptospira spp. Genomic DNA
Expression Library ... 151 4.2.2(a) Titration of Genomic DNA Expession Library &
Estimation of Its Coverage on Genome ... 151 4.2.2(b) Optimization of Phage Dilution ... 154 4.2.2(c) Optimization of Conjugate Dilution ... 154
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4.2.3 Identification of Immunoreactive Lambda Phage Clones to
Leptospirosis Patients ... 156 4.2.3(a) Primary and Secondary Immunoscreening ... 156 4.2.3(b) Tertiary Immmunoscreening (Diagnostic
Potential of In Vivo Induced Antigens) ... 159 4.3 Production of Recombinant Leptospiral In Vivo Induced Antigen and
Other “Reported” Serodiagnostic Markers for Leptospirosis ... 166 4.3.1 In Silico Characterization of Gene Insert in S8A1 Phage
Clone ... 166 4.3.1(a) Data Mining and Biophysical Properties ... 166 4.3.1(b) Similarity Search for LA_0153 Gene Sequence in
NCBI Protein Databases ... 171 4.3.1(c) Multiple Sequence Alignment of LA_0153
Protein Homologues ... 173 4.3.1(d) Phylogenetic Analysis of LA_0153 Protein
Homologues ... 180 4.3.1(e) Design and Construction of Prokaryotic
Expression Vector for LepS8A1FL, Its Truncated Derivatives and Other Reported Leptospira
Protein Antigens ... 183 4.3.2 Expression and Purification of Recombinant LepS8A1FL
Protein, Its Truncated Derivatives and Reported Leptospira
Protein Antigens ... 193 4.3.2(a) Expression Profile of Recombinant LepS8A1FL
Protein, Its Truncated Derivatives and Reported
Leptospira Protein Antigens ... 193 4.3.2(b) Purification of Recombinant LepS8A1 Protein,
Its Truncated Derivatives and Reported
Leptospira Protein Antigens ... 198 4.3.2(c) Identification of Purified Proteins ... 201 4.3.2(d) Isoelectric Point Determination for S8A1 Protein ... 208 4.4 Diagnostic Performance Evaluation of Recombinant LepS8A1FL and
other Leptospira Protein Antigens ... 211 4.4.1 Determination of Recombinant LepS8A1FL, Its Truncated
Derivatives and Other Leptospira Protein Antigens
Diagnostic Value in Western Blot ... 211
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4.4.1(a) Optimization of Western Blot Condition ... 211 4.4.1(b) Diagnostic Performance Evaluation of
Recombinant LepS8A1FL and other Leptospira
Protein Antigens in Western Blot ... 213 4.4.1(c) Use of Dual Assays for Diagnosis of
Leptospirosis: Recombinant LepS8A1FL IgM
Western Blot and Commercial Rapid Test ... 220 4.4.2 Determination of Recombinant LepS8A1FL and Its
Truncated Derivatives Diagnostic Value in IgM Enzyme-
Linked Immunosorbent Assay (ELISA) ... 223 4.4.2(a) Optimization of IgM ELISA Condition... 223 4.4.2(b) Evaluation of Recombinant LepS8A1FL,
LepS8A124 and LepS8A134 IgM ELISA
Performance ... 225 4.4.2(c) Use of Dual Assays for Diagnosis of
Leptospirosis: Recombinant LepS8A1FL IgM
ELISA and Commercial Rapid Test ... 228 4.5 Development of A Rapid Antibody Detection Test for Acute
Leptospirosis in IgM LFD Format Using Recombinant LepS8A1FL
Protein Antigen ... 231 4.5.1 Refolding of Denatured Recombinant LepS8A1FL Protein for
Use as Antigen in IgM LFD Assay ... 231 4.5.2 Development of A Rapid Recombinant LepS8A1FL IgM
LFD Assay for Serodiagnosis of Acute Leptospirosis ... 233 4.5.3 Use of Dual Assays for Diagnosis of Leptospirosis:
Recombinant LepS8A1FL IgM LFD Assay and Commercial
Rapid Test ... 236 4.6 Development of A Rapid Antigen Detection Test for Acute
Leptospirosis in LFD Format ... 239 4.6.1 Production and Immunoreactivity of Rabbit Polyclonal
Antibodies Against Leptospiral Proteins ... 239 4.6.1(a) Antibody Titer Determination ... 241 4.6.2 Immunodetection of Leptospira Endogenous LA_0153
Protein ... 245 4.6.3 Production of Gold Nanoparticle-Conjugated Anti-
Leptospiral-Protein IgG ... 247
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4.6.4 Development of A Rapid Antigen Detection Test for
Serodiagnosis of Acute Leptospirosis ... 250 4.6.4(a) Determination of The Best Assay Format for
Detection of Leptospira spp. ... 250 4.6.4(b) Reactivity, Limit of Detection and Specificity of
Antigen Detection LFD Assay for Leptospirosis ... 252 4.6.4(c) Diagnostic Performance Evaluation of Antigen
Detection LFD Assay ... 255 4.6.4(d) Use of Dual Assays for Diagnosis of
Leptospirosis: Leptospira Antigen Detection Lateral Flow Dipstick Test and Commercial
Rapid Test ... 258 4.6.5 Development of A Rapid Urine Antigen Detection Test for
Diagnosis of Acute Leptospirosis ... 261 4.7 Determination of The Best Dual Assays for Acute Leptospirosis
Diagnosis – A Wrap-Up ... 265
CHAPTER FIVE: DISCUSSION ... 268 5.1 Low Diagnostic Performance of Commercial Leptospirosis Rapid
Diagnostic Kits in Detecting Acute Phase Leptospirosis Serum ... 270 5.2 In Vivo Induced Antigen Technology Identified Six Upregulated
Leptospiral Genes In Vivo ... 275 5.3 In Silico Finding of LA_0153 Homologues in Leptospira Species ... 284 5.4 Endogenous Expression of LA_0153 Protein in Pathogenic Leptospira ... 289 5.5 Analysis and Improvement of LepS8A1FL and Other Leptospiral
antigens for High Yield Heterologous Production in Prokaryotic
Expression System ... 290 5.6 LepS8A1FL Protein as A Biomarker for Detection of Early Acute
Leptospirosis ... 295 5.7 Antigen Detection Test for Diagnosis of Acute Phase Leptospirosis ... 299
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CHAPTER SIX: CONCLUSION ... 305 6.1 Limitations of The Study and Suggestions for Future Studies ... 308
REFERENCES ... 310 APPENDICES
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LIST OF TABLES
Page Table 2.1 Scoring system of Faine’s Criteria and its modified versions ... 35 Table 2.2 Examples of commercial leptospirosis diagnosis kits in the
market ... 55 Table 3.1 Leptospira spp. serovars used in this study ... 68 Table 3.2 Escherichia coli strains and control bacteria in this study ... 69 Table 3.3 Recombinant plasmids and oligonucleotide DNA used in this
study ... 70 Table 3.4 Kits used in this study ... 71 Table 3.5 Bioinformatics software and tools in this study ... 72 Table 3.6 Recipe for lysis and washing buffers for native purification of
proteins in this study ... 80 Table 3.7 Reference Leptospira serovars used in Microscopic
Agglutination Test panel ... 89 Table 3.8 Schematic diagram to show rating system for potential lambda
phage clones identified from immunoscreening the Leptospira
genomic DNA expression library ... 106 Table 3.9 Amino acid residue colored by GeneDoc software ... 111 Table 3.10 Composition of SDS-polyarcylamide gel ... 117 Table 4.1 Diagnostic sensitivity and specificity of Leptorapide and
VISITECT-LEPTO for anti-leptospiral antibodiesdetection ... 141 Table 4.2 Performance evaluation of Leptorapide and VISITECT-
LEPTO with patient control sera ... 144 Table 4.3 List of patient serum samples used for immunoscreening of
leptospiral genomic DNA expression library ... 146 Table 4.4 Average plaque counts of leptospiral genomic DNA
expression library at different dilutions ... 153 Table 4.5 Number of phage clone candidate and their respective rating
score ... 158 Table 4.6 Leptospira in vivo induced genes identified by IVIAT ... 160
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Table 4.7 Seroreactivity of phage clones carrying leptospiral in vivo
induced antigens ... 164 Table 4.8 Alignment between LA_0153 gene product and NCBI non-
redundant protein database ... 172 Table 4.9 Rare codon analysis of genes used in this study ... 184 Table 4.10 Prediction of signal peptides in leptospiral proteins ... 191 Table 4.11 Optimization of recombinant Leptospira protein antigen
amount in western blot ... 212 Table 4.12 Diagnostic performance of recombinant LepS8A1FL and its
truncated derivatives by IgM western blot ... 215 Table 4.13 Detailed diagnostic performance analysis of recombinant
LepS8A1FL and its truncated derivatives by IgM western blot .... 216 Table 4.14 Diagnostic performance of recombinant LigA and LipL41 by
IgM western blot ... 218 Table 4.15 Detailed diagnostic performance analysis of recombinant
LigA and LipL41 by IgM western blot... 219 Table 4.16 Analysis of results of recombinant LepS8A1FL IgM western
blot when combined with results of commercial leptospirosis
rapid diagnostic kits ... 222 Table 4.17 Diagnostic performance of recombinant LepS8A1FL and its
truncated derivatives by in house IgM ELISAs ... 226 Table 4.18 Detailed diagnostic performance analysis of recombinant
LepS8A1FL and its truncated derivatives by in house IgM
ELISAs ... 227 Table 4.19 Analysis of results of recombinant LepS8A1FL IgM Enzyme-
Linked Immunosorbent Assay when combined with results of
commercial leptospirosis rapid diagnostic kits ... 229 Table 4.20 Diagnostic performance of LepS8A1FL IgM dipstick test for
acute leptospirosis ... 234 Table 4.21 Detailed diagnostic performance analysis of LepS8A1 IgM
dipstick test for acute leptospirosis ... 235 Table 4.22 Analysis of results of recombinant LepS8A1FL IgM dipstick
test when combined with results of commercial leptospirosis
rapid diagnostic kits ... 237 Table 4.23 Checkerboard optimization of ELISA for rabbit antiserum
titer determination ... 243
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Table 4.24 Summary of experiment for indirect evidence of LA_0153 protein expression in pathogenic Leptospira grown in vitro by
using western blot ... 248 Table 4.25 Diagnostic performance of antigen detection lateral flow
dipstick assay for acute leptospirosis ... 256 Table 4.26 Detailed diagnostic performance analysis of antigen detection
lateral flow dipstick assay for acute leptospirosis ... 257 Table 4.27 Analysis of results of Leptospira antigen detection lateral flow
dipstick assay when combined with results of commercial
leptospirosis rapid diagnostic kits ... 260 Table 4.28 Diagnostic performance summary and combinative analysis
of all diagnostic tests developed and/or evaluated in this study.
Outstanding performances are highlighted ... 266
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LIST OF FIGURES
Page Figure 2.1 Distribution and leptospirosis burden illustrated in Disability
Adjusted Life Years (DALYs)/100,000 population per year ... 5
Figure 2.2 Trends on leptospirosis incidence and death cases in Malaysia between 2004 to July 2015 ... 7
Figure 2.3 Phylogenetic analysis of Leptospira spp. 16S rRNA and classification of Leptospira based on its pathogenicity ... 14
Figure 2.4 Morphology of Leptospira interrogans ... 18
Figure 2.5 Schematic diagram of Leptospira spp. cell wall ... 19
Figure 2.6 Illustration of leptospiral motility ... 24
Figure 2.7 Common clinical complication of severe leptospirsosis ... 31
Figure 2.8 Bacterial load, antibody kinetic and the relevant diagnosis methods for leptospirosis ... 37
Figure 2.9 Silver impregnation stain for visualization of pathogenic Leptospira cell in rat kidney ... 41
Figure 2.10 An overview of In-Vivo Induced Antigen Technology procedure ... 58
Figure 3.1 Experimental overview of the study ... 63
Figure 3.2 Type and categorization of serum used in this study ... 90
Figure 3.3 Result interpretation of commercial leptospirosis rapid diagnosis test kits used in this study ... 93
Figure 3.4 Petroff-Hausser counting chamber ... 136
Figure 4.1 Flowchart showing types of serum samples used in evaluation of leptospirosis diagnostic tests, Leptorapide and VISITECT- LEPTO ... 140
Figure 4.2 Evaluation of serum pre-absorption efficiency with ELISA ... 148
Figure 4.3 Evaluation of absorption efficiency after sequential steps of serum absorption ... 149
Figure 4.4 Determination of minimum clone number required for a Leptospira genomic DNA library and fold coverage of the constructed leptospiral genomic DNA expression library. ... 152
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Figure 4.5 Optimization of secondary antibody dilution for
immunoscreening ... 155 Figure 4.6 Representative duplicate immunoscreening blot with pre-
absorbed pooled challenge and leptospirosis serum samples ... 157 Figure 4.7 Serum absorption with three kinds of E. coli and leptospiral
protein preparations ... 161 Figure 4.8 Evaluation of diagnostic potential of S8A1 phage clone... 165 Figure 4.9 Homologous region of S8A1 phage clone insert in L.
interrogans serovars Lai reference genome ... 167 Figure 4.10 Multiple sequence alignment of S8A1 phage insert, LA_0153
gene and their homologous region in the L. interrogans
serovars Lai str. 56601 reference genome ... 168 Figure 4.11 Nucleotide and amino acid sequence of LepS8A1 ... 170 Figure 4.12 Multiple sequence alignment of LA_0153 homologues in
different Leptospira species ... 174 Figure 4.13 Multiple sequence alignment of LA_0153 homologues in
different Leptospira species ... 177 Figure 4.14 Phylogeny tree of LA_0153 protein homologues in Leptospira
species ... 181 Figure 4.15 Native and codon-optimized DNA sequence of LepS8A1 and
their deduced amino acid ... 186 Figure 4.16 Linear B cell antibody epitope prediction for LepS8A1FL
protein ... 187 Figure 4.17 Superimposition of LepS8A1FL epitopes predicted by
multiple methods ... 188 Figure 4.18 Map of pET-28a(+) expression vector showing its features and
location of recombinant LepS8A1FL, LepS8A124 or LepS8A134 open reading frame that are embedded in the
vector sequence ... 190 Figure 4.19 Expression and detection of recombinant LepS8A1FL and its
truncated derivatives in Escherichia coli BL21(DE3) ... 194 Figure 4.20 Solubility study of recombinant LepS8A1FL ... 195 Figure 4.21 Expression analysis of reported recombinant leptospiral
proteins ... 197
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Figure 4.22 Denaturing purification of recombinant LepS8A1FL and its
truncated derivatives ... 199 Figure 4.23 Native purification of recombinant LepS8A1FL and
LepS8A134 ... 200 Figure 4.24 Denaturing purification of recombinant LigA, LipL32,
LipL41 and OmpL1 ... 202 Figure 4.25 Western blot verification of purified recombinant Leptospira
protein antigens with anti-His antibody ... 204 Figure 4.26 MALDI-TOF/TOF protein summary report for recombinant
LepS8A1FL protein ... 206 Figure 4.27 MALDI-TOF/TOF peptide summary report for recombinant
LepS8AlFL protein. ... 207 Figure 4.28 OFFGEL electrophoresis of recombinant LepS8AlFL and
LepS8A134 ... 209 Figure 4.29 Representative result of an IgM western blot showing
immunoreactivity between human sample IgM and
recombinant LepS8A1FL as well as its truncated derivatives. ... 214 Figure 4.30 Qualitative analysis of refolded recombinant LepS8A1FL
protein profile by SDS-PAGE ... 232 Figure 4.31 Reactivity of rabbit hyperimmune serum with Leptospira
protein antigens in western blot ... 240 Figure 4.32 Cross reactivity between recombinant LepS8A1FL protein
antigen and its truncated derivatives with their respective
rabbit antiserum ... 242 Figure 4.33 Determination of rabbit antiserum titer by ELISA. With
optimized parameter, ELISA was performed with different
dilution of rabbit antiserum ... 244 Figure 4.34 Direct and indirect immunodetection of endogenous LA_0153
protein in pathogenic Leptospira spp ... 246 Figure 4.35 Representative protein profile of rabbit serum and
Immunoglobulin G ... 249 Figure 4.36 Determination of best assay format for detection of Leptospira
using dot dipstick format ... 251 Figure 4.37 Reactivity of leptospirosis antigen detection lateral flow
dipstick assay towards bacteria ... 253
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Figure 4.38 Performance evaluation of the leptospirosis antigen detection
lateral flow dipstick assay ... 254 Figure 4.39 Reactivity of leptospirosis urine antigen detection lateral flow
dipstick assay towards bacteria ... 262 Figure 4.40 Performance evaluation of the leptospirosis urine antigen
detection lateral flow dipstick assay ... 264
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LIST OF ABBREVIATIONS ABTS 2,2’-Azino-d-[3-ethylbenthiazoline sulfonate]
APS ammonium persulfate
BLAST Basic Local Alignment Search Tool
bp base pair
BSA bovine serum albumin CAI codon adaptation index
COV cut-off value
CSF cerebrospinal fluid DMSO dimethyl sulfoxide DNA deoxyribonucleic acid
e.g. Exempli gratia "for example"
ECL enhanced chemiluminescence EDTA ethylenediaminetetraacetic acid ELISA enzyme-linked immunosorbent assay EMJH Ellinghausen-McCullough-Johnson-Harris
g gravity force
His histidine
HRP horseradish peroxidase
i.e. that is
IgG immunoglobulin G
IgM immunoglobulin M
IMR Institute for Medical Research
IPTG isopropyl-beta-D-thiogalactopyranoside IVIAT in-vivo Induced Antigen Technology
Kb kilo base pair
kDa kilodalton
LFD lateral flow dipstick LL leptospiral lysates
LPS lipopolysaccharide
MALDI- TOF/TOF
matrix-assisted laser desorption/ionization-time of flight/time of flight
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MOH Ministry of Health
NC nitrocellulose membrane Ni-NTA nickel-nitrilotriacetic acid NPV negative predictive value
OD optical density
OM outer membrane
ORF open reading frame PBS phosphate buffered saline PCR polymerase chain reaction pfu plaque forming unit pI isoelectric point
PI protease inhibitor
PPV positive predictive value
RC DCTM reducing compatible detergent compatible rpm rotation per minute
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
TB terrific broth
TBS tris buffered saline
TEMED tetramethylethylenediamine
UV ultra violet
V Volt
X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
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PROTEIN ARUHAN IN-VIVO Leptospira DAN APLIKASINYA DALAM PEMBANGUNAN UJIAN PENGESANAN ANTIBODI DAN ANTIGEN
UNTUK PENYAKIT LEPTOSPIROSIS AKUT
ABSTRAK
Leptospirosis yang disebabkan oleh Leptospira spp. patogenik merupakan ancaman kesihatan sedunia yang muncul semula. Justeru, adalah penting untuk mengenalpasti penanda diagnostik baru dan menyelidik potensi penggunaannya dalam asai pengesanan antibodi dan antigen bagi diagnosis leptospirosis akut. Dengan menggunakan panel sampel serum daripada leptospirosis fasa akut (Kumpulan I) dan campuran (Kumpulan II), kajian ini dimulakan dengan menilai prestasi dua kit diagnostik pantas leptospirosis, iaitu Leptorapide dan VISITECT-LEPTO, yang lazim digunakan di Malaysia. Kedua-dua kit ujian tersebut menunjukkan sensitiviti diagnostik yang rendah (≤34%) terhadap sampel serum fasa akut, tetapi sensitiviti yang lebih baik terhadap sampel fasa campuran. Sampel serum yang terpilih dari Kumpulan I digunakan untuk mengenalpasti penanda diagnostik baru daripada perpustakaan ekspresi DNA genomik Leptospira dengan menggunakan teknologi antigen aruhan in-vivo (IVIAT). Klon faj S8A1 telah dikenalpasti dan pecahan gen- nya (dinamakan sebagai LepS8A1FL) telah diklonkan ke dalam plasmid rekombinan.
Protein tersebut seterusnya diekspres dalam sistem ekspresi protein Escherichia coli dan ditulen dengan menggunakan “Immobilized Metal Affinity Chromatography”.
Dua terbitan terpenggal LepS8A1FL (dinamakan sebagai LepS8A124 and LepS8A134) dan penanda-penanda diagnostik leptospirosis yang pernah dilaporkan (dinamakan sebagai LigA, LipL41, OmpL1 dan LipL32) turut dihasilkan dengan kaedah yang ternyata di atas. Dalam blot western Immunoglobulin M (IgM),
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LepS8A1FL menunjukkan prestasi yang memuaskan dengan sensitiviti dan spesifisiti 75%. Ia mengungguli LipL41 dan LigA yang pernah dilapor dalam mengesan leptospirosis akut. Walau bagaimanapun, protein tersebut menunjukkan nilai diagnostik sederhana dalam format asai “Enzyme-Linked Immunosorbent” (ELISA) IgM. Satu asai dipstik aliran sisi (LFD) IgM yang menggunakan LepS8A1FL seterusnya telah dibangunkan, dan masing-masing menunjukkan sensitiviti dan spesifisiti diagnostik sebanyak 65.7% dan 63.2%. Kajian ini diteruskan dengan ujian pengesanan antigen untuk leptospirosis akut. Untuk tujuan ini, antibodi poliklonal arnab terhadap pecahan sel kasar Leptospira dan kesemua protein rekombinan leptospiral tersebut telah dihasilkan. Asai LFD yang menggunakan antibodi anti- pecahan leptospiral sebagai kedua-dua antibodi tangkapan dan antibodi berkonjugat emas mengungguli kombinasi antibodi yang lain. Ia menunjukkan limit pengesanan yang tinggi, spesifisiti yang baik serta reaktiviti yang luas. Ujian tersebut menunjukkan sensitiviti dan spesifisiti diagnostik masing-masing 57.4% dan 87.2%. Ujian pengesanan antigen tersebut juga mengesan antigen leptospiral di dalam air kencing manusia. Di samping itu, dengan menggabungkan keputusan ELISA IgM LepS8A1FL dan asai LFD pengesanan antigen tersebut, sensitiviti diagnostik 86.2% dan spesifisiti diagnostik 73.7% tercapai. Secara kesimpulannya, kedua-dua ujian pengesanan antibodi dan antigen dalam kajian ini patut dibangunkan selanjutnya untuk digunakan dalam diagnosis leptospirosis akut pada manusia.
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LEPTOSPIRAL PROTEINS INDUCED IN-VIVO AND ITS APPLICATION IN THE DEVELOPMENT OF ANTIBODY AND ANTIGEN DETECTION
TESTS FOR ACUTE LEPTOSPIROSIS
ABSTRACT
Leptospirosis, caused by pathogenic Leptospira spp., is a re-emerging global health threat. Therefore, it is pertinent to identify novel diagnostic marker and investigate its potential use in antibody and antigen detection assays for detection of acute leptospirosis. Using panels of serum samples from acute (Group I) and mixed (Group II) phase leptospirosis, the present study was initiated by evaluating performance of two leptospirosis rapid diagnostic kits, namely Leptorapide and VISITECT-LEPTO, that were commonly used in Malaysia. Both test kits showed low diagnostic sensitivity (≤34%) with the acute phase serum samples, but better sensitivity with the mixed phase samples. Selected serum samples from Group I were used to identify novel diagnostic marker(s) from Leptospira genomic DNA expression library using In-vivo Induced Antigen Technology. A phage clone, S8A1, was identified and its gene fragment (named as LepS8A1FL) was cloned into recombinant plasmids. The protein was then expressed in Escherichia coli protein expression system and purified using Immobilized Metal Affinity Chromatography. Two truncated derivatives of the LepS8A1FL (namely LepS8A124 and LepS8A134) and previously reported diagnostic markers for leptospirosis (namely LigA, LipL41, OmpL1 and LipL32) were produced with the methods described above. In Immunoglobulin M (IgM) western blot, LepS8A1FL showed a satisfactory performance with 75.0% sensitivity and specificity. It outperformed the reported LipL41 and LigA in detecting acute leptospirosis. In an IgM Enzyme-Linked
xxvii
Immunosorbent Assay (ELISA) format, the protein, however, has moderate diagnostic value. An IgM lateral flow dipstick assay (LFD) using LepS8A1FL was subsequently developed and demonstrated diagnostic sensitivity and specificity of 65.7% and 63.2%, respectively. The present study also pursued an antigen detection test for acute leptospirosis. For this purpose, rabbit polyclonal antibodies against Leptospira crude cell lysate and all of the above leptospiral recombinant proteins were produced. The LFD assay which used anti-leptospiral lysate antibody as both immobilized and gold- labelled antibody was superior to other antibody combinations. It showed high limit of detection, good specificity and broad reactivity. The test demonstrated diagnostic sensitivity and specificity of 57.4% and 87.2%, respectively. The antigen detection test also detected leptospiral antigen in human urine. Meanwhile, by combining the results of LepS8A1FL IgM ELISA and the antigen detection LFD test, a diagnostic sensitivity of 86.2% and diagnostic specificity of 73.7% was achieved. In conclusion, both antibody and antigen detection tests in this study merit further development for use in diagnosing acute leptospirosis in human.
1 1.0 Introduction
Leptospirosis is a bacterial infection caused by pathogenic species of Leptospira. The disease is most prevalent in tropical regions because of the warm and high humidity atmosphere (Picardeau, 2013). The pathogen may potentially infect all mammals, including human. Depending on the animals and the infecting serovars, the disease may cause a wide range of manifestation to animals, including asymptomatic, acute, chronic symptoms and carrier stage (Jobbins and Alexander, 2015; Adler, 2014). Upon acquiring the infection, the chronic-infected or carrier animals can further contaminate the surrounding environment by passing urine containing the bacteria into soil and fresh water streams. Thus, other potential hosts who live in the epidemic area may be exposed to the bacteria and further propagate the disease (Subharat et al., 2011).
Human is an accidental host for the pathogen. Leptospirosis cases are estimated to be approximately one million and 58,900 deaths per annum (Costa et al., 2015).
This represents 14.77 cases and 0.84 deaths per 100,000 population, respectively.
Human-to-human transmission is practically non-existent. The disease is transmitted to human upon contact with water contaminated by animal hosts as described above.
The bacteria enters human body via the breached skins and mucous membranes (Picardeau, 2017). Four major risk factors expose human to pathogenic Leptospira: i) recreational water activities, ii) watery-associated occupations e.g. paddy and poultry farmers as well as workers in slaughterhouse, iii) post-natural disasters e.g. flood and typhoon, and iv) poor hygiene.
Leptospirosis is a biphasic disease (Gasem et al., 2009). Following an incubation period of 5-14 days, a patient is in acute (leptospiraemic) phase and
CHAPTER ONE INTRODUCTION
2
develops symptoms such as high fever, headache and myalgia, which are similar to other febrile illnesses such as dengue and malaria. If left untreated, the pathogen may infect vital organs during convalescent phase and cause mortality to a patient.
Nevertheless, the disease can be treated effectively by administrating appropriate antibiotic regimen (Phimda et al., 2007). Hence, a good clinical management highly depends on correct identification of the disease. Due to unspecific clinical symptoms of acute leptospirosis, laboratory diagnosis become a critical tool to detect the disease and further support clinical decision of a suspected patient (McBride et al., 2007).
Many diagnostic tools to detect anti-leptospiral antibody are readily available in the market. However, there is a big room to improve leptospirosis diagnosis because performance of most kits are poor in detecting acute phase of leptospirosis due to the absence or low anti-leptospiral antibody in the early disease stage (Bajani et al., 2003).
Currently, most of the serological tests for leptospirosis used crude cell proteins from the non-pathogenic L. biflexa as the antigens. The strategy is relatively straightforward and convenient for manufacturers. However, one of its major drawback is its inconsistency of diagnostic performance in different countries. This is because the predominant Leptospira serovar varies between countries. As a result, the anti-leptospiral antibody developed by a patient, which is serovar specific, may not be efficiently detected by those serological tests (Blacksell et al., 2006). To overcome this limitation, it is necessary to identify diagnostic marker(s) that is conserved across different Leptospira serovars. To date, several protein antigens, such as LipL21, LipL32 and LipL41 have been identified (Natarajaseenivasan et al., 2008; Boonyod et al., 2005; Cullen et al., 2003). Yet, their performance differs particularly in detecting the IgM antibody in acute leptospirosis sera (Toyokawa et al., 2011). As such, new approaches should be implemented to identify novel antigenic markers for
3
serodiagnosis of leptospirosis. It is known that bacterial proteins which are highly expressed during infection event (also known as in vivo induced proteins) may be incorporated into diagnostic tool as antigens to detect the antibodies raised in a patient (Cao et al., 2004). In Vivo Induced Antigen Technology (IVIAT) is a method to identify these diagnostic markers by immunoscreening genomic or complementary DNA library of a particular microorganism without the need to use an animal model (Hu et al., 2014). The technology facilitates identification of immunogenic leptospiral protein antigens that are expressed during infection event. Previously, IVIAT has been used to discover novel diagnostic markers from several pathogens such as Mycobacterium tuberculosis, Vibrio cholera and Bacillus anthracis (Kumar et al., 2011; Rollins et al., 2008; Hang et al., 2003). Based on the results of the above studies, IVIAT-identified protein(s) may be useful for IgM detection of leptospirosis in human.
On the other hand, it is known that leptospiral antigens are circulating in a leptospirosis patient during acute phase (Picardeau, 2013). Thence, detection of the circulating antigens demonstrates direct evidence of acute leptospirosis. Unlike IgM detection tests, antigen detection test is not in the market. As a result, the current study pursues this approach by using antibodies to selected leptospiral antigens, such as LigA, LipL32, LipL41 and OmpL1, since these proteins are reported to be abundant in the bacteria (Malmstrom et al., 2009). In addition to that, this study was also designed to detect whole cell antigens of Leptospira spp. as an approach to include undefined circulating leptospiral antigens. This antigen detection strategy may complement with antibody detection test described above for improved diagnosis of acute leptospirosis in human.
4 2.0 Literature Review
2.1 World Epidemiology
Due to Leptospira spp. preference to proliferate in hot and wet atmosphere, majority of leptospirosis incidence occurred in tropical countries located between tropic of Capricorn and Cancer (Figure 2.1). In tropical countries, its annual incidence rate exceeds 10 cases/100,000 population which was higher than the reported 0.1-1 case/100,000 population in temperate climates (Picardeau, 2013). Highest morbidity rate was reported at Oceania (150.68/100,000 population), followed by South East Asia (55.54/100,000 population), Caribbean (50.68/100,000) and East Sub-Saharan Africa region (25.65/100,000) (Costa et al., 2015). Of note, small tropical countries or islands in these regions are highly endemic for leptospirosis. A work by Pappas et al.
(2008) revealed Seychelles (43.2/100,000 population) to be the country in the world with highest incidence rate, followed by Trinidad and Tobago (12.0/100,000 population) and Barbados (10.0/100,000 population). However, the actual prevalence might be more serious than those reported because data from developing countries are normally under-estimated and less reliable (Pappas et al., 2008). Considering low socioeconomic status of the regions, the disease might be under-recognized as a potential public health threat (Schneider et al., 2013). In addition to that, long heavy rainfall season in tropical climate further promotes the disease incidence (Ko et al., 1999).
CHAPTER TWO LITERATURE REVIEW
5
Figure 2.1 Distribution and leptospirosis burden illustrated in Disability Adjusted Life Years (DALYs)/100,000 population per year. One DALYs unit represents one year which a healthy individual lost due to a circumstance, e.g. disease and disability. Reprinted from Torgerson, P.
R., et al. (2015).
Tropic of Cancer
Tropic of Capricorn Equator
6 2.1.1 Leptospirosis in Malaysia
Leptospirosis is an endemic disease in Malaysia. The first recorded human leptospirosis study in Malaysia was performed on rural inhabitants and rubber plantation workers by Fletcher (1928). As an endemic country for leptospirosis, the average morbidity rate was estimated to range between 1-10/100,000 population (Lim, 2011). Figure 2.2 illustrates trends of leptospirosis incidence and death cases for the past 11 years. A nine-year study (2004-2012) conducted by Benacer et al. (2016) demonstrated an average annual incidence rate of 4.83/100,000 population. However, the annual incidence rate markedly increased year to year, noting from 0.97 (2004) to 12.47 (2012) cases/100,000 population throughout the study. Abdul Wahab (2015) reported that the highest leptospirosis incidence rate (25.5 cases/100,000 populations) and mortality cases (92 cases) in Malaysia occurred in 2014. A spiked increase over 60% of cases reported compared to its preceding year has been linked to heavy rainfall and flood that happened in many states of Malaysia during the year (Garba et al., 2017). Continuous increase in reported leptospirosis cases was correlated to the fact that leptospirosis was instated as a national notifiable disease since 9th December 2010 under Prevention and Control of Infectious Disease Act 1988. Following year, an official guideline by Ministry of Health (MOH) Malaysia regarding diagnosis, management, prevention and control of the disease was published. These approaches commit better surveillance and monitoring by health institution and government.
Particularly, it raises awareness of the disease among clinical practitioners, which contributed to better prognosis and early treatment initiation to the suspected patients.
In Malaysia, male gender constituted 78.7% of total leptospirosis cases in between 2004-2012 (Benacer et al., 2016). This contributed a male-to-female ratio of 3.69:1. The phenomenon of male outnumbered female patients is common in
7
Figure 2.2 Trends on leptospirosis incidence and death cases in Malaysia between 2004 to July 2015. Data adapted from Abdul Wahab (2015).
263 378 527 949 1263 1418
1976 2268 3665
4457 7806
5307
20 20 22 22
47 62
69 55
48 71
92
30
0 10 20 30 40 50 60 70 80 90 100
0 1000 2000 3000 4000 5000 6000 7000 8000 9000
2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015*
Death cases
Total Cases
Year
Total cases Death cases
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leptospirosis worldwide and has been associated with high risk occupation and recreational activities predominated by male (Costa et al., 2014; Felzemburgh et al., 2014). Malaysian patient demography reported the mean (±SD) age of leptospirosis patients was 33.79 (±17.55) and median of 31 years old (Benacer et al., 2016).
Malaysian within age group of 30-39 years old has the highest morbidity rate (16.21 cases/100,000 population). Constitution of age group 30-39 years old as the major group for leptospirosis was similarly observed in different epidemiological settings (Costa et al., 2015; Goris et al., 2013a). Compared to school children and adolescent, it was predicted that middle age adult possessed more mobility which lead to higher exposure risk to the disease (Benacer et al., 2016).
Leptospirosis risk is frequently present during rainfall and flood season in developing countries, whereas water recreational activities are more relevant in developed countries (Mwachui et al., 2015). In Malaysia, annual reported leptospirosis cases similarly follow the seasonality pattern. A spiked case number was reported in October-March in peninsular Malaysia and in October-February in east Malaysia (Benacer et al., 2016). This is in concordance with wet season in the country.
Weinberger et al. (2014) claimed that rainfall flush the Leptospira spp. bacteria which typically survive in wet soils into water bodies. As such, human at watery grounds is more prone to the infection. However, excessive volume of rainfall provides dilution effect to the bacteria load, which inversely present as a protective factor to the flood victim (Suwanpakdee et al., 2015).
Thayaparan et al. (2015) demonstrated a seroprevalence of 35.9% among villagers (n=198) in periurban area of Kuching, Sarawak. Villagers who work around forest and involve in national service exhibit seroprevalence of more than 50%.
Meanwhile, plantation workers in Johor and Melaka recorded a seroprevalence of
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28.6% (Janudin et al., 2016). Leptospira serovar Lepto 175 Sarawak was the predominant serovar in these oil palm plantations, contributing to 62% among the seropositive workers (Janudin et al., 2016). A cross-sectional study of leptospirosis seroprevalence among 999 febrile patients in ten Kelantan government hospitals showed higher leptospirosis seroprevalence among patients from high-risk occupational group, e.g. outdoor worker, agriculture worker and military (Rafizah et al., 2013a). In addition to that, Rafizah et al. (2013b) reported that patient with exposure to recreational activity has 2.4 times higher risk for leptospirosis.
2.1.2 Outbreaks and Case Reports in Malaysia
As an endemic country, leptospirosis outbreaks happen intermittently in Malaysia. The most recent scientific literature about leptospirosis outbreak in Malaysia was dated 2012. Leptospirosis outbreak was reported in Pauh, Perlis among family members after fisheries activity at a swamp in Kampung Padang Telela (Baharudin et al., 2012). The swamp was an abandoned paddy field and has been neglected for long time. After approximately two weeks, eight out of 28 of the participants involved showing common febrile symptoms such as fever, headache, vomiting and muscle pain.
Serology test for the presence of leptospiral IgM using immunochromatographic test (VISITECT-LEPTO, Omega-Diagnostic, UK) demonstrated six samples to be positive. Most of the samples were then further confirmed to be positive by MAT and/or Polymerase Chain Reaction (PCR) at Institute for Medical Research (IMR) Malaysia. Pathogenic Leptospira spp. DNA was identified in seven out of eight water samples from the swamp. Hence, the incident was categorized as a point-source outbreak.
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On 26th June 2010, a search and rescue operation on a drowned young man was conducted in Lubuk Yu, Pahang. Lubuk Yu is a recreational forest with river and waterfall. Among 153 people who participated the rescue, 21 of them developed fever
≥ 38oC seven days post-operation (IQR 1-13 days) (Sapian et al., 2012). Ten serum samples of the patients were cultured-positive for Burkholderia pseudomallei. Out of which four serum samples were positive for leptospirosis by PCR, suggesting co- infection with Leptospira spp. High fatality rate of 38% (8/21) was reported in this outbreak. In detail, fatality rate for melioidosis alone and melioidosis-leptospirosis co- infection were 66.7% (4 out of 6) and 75% (3 out of 4), respectively. All water samples (n=6) and two out of four soil samples collected on site of operation showed positive for Leptospira spp. This has been linked to heavy rainfall during first two days of the operation which might flush the bacteria onto the soil surface and into the river.
The first international leptospirosis outbreak happened in Sabah (Sejvar et al., 2003). During 21st August to 1st September 2000, a number of 304 athletes participated in Eco-Challenge Sabah multisport endurance race. Fifteen days (range one to 24 days) after the race, 80 out of 189 athletes showed common febrile symptoms, including fever, headache, chills, diarrhea and muscle aches. In addition to that, 40 athletes showed conjunctival suffusion which is a representative symptom for leptospirosis. A total of 29 case-patients were admitted to hospital. All the patients recovered and no death case was reported. Serological test showed positive reaction in 68% (26/38) of the serum samples. Multivariate stepwise logistic regression demonstrated that swimming in Segama River is the main risk factor contributed to this point-source outbreak. Abrasions and cuts on the athletes in jungle trekking preceding to swimming in the river increased the risk of exposure to Leptospira spp.
11 2.2 Classification and Typing of Leptospira 2.2.1 Taxonomy
Leptospires belong to family Leptospiraceae in the phylum of Spirochetes. The phylum members consisted of mammalian pathogens which cause a vast array of serious diseases, particularly in human. Besides leptospirosis, some notorious human diseases caused by the spirochetes members are syphilis (Treponema pallidum), Lyme disease (B. burgdorferi), relapsing fever (Borrelia spp.), yaws (T. pallidum subsp.
Pertenue), pinta (T. carateum) and periodontal disease (Treponema spp.). In general, spiral shapes and endoflagella motility are the hallmarks of spirochetes. They demonstrated morphology of long, thin bacteria with flat-waves, helices or irregular shape under the microscope (Wolgemuth, 2015). These special modes of propulsion and morphology indeed represents virulence factors of spirochetes. However, in year 2012 a novel genus, namely Sphaerochaeta, was included as a member of phylum Spirochaetes (Caro-Quintero et al., 2012). It implies an exception to the morphology hallmark of spirochetes as the species are non-motile and sphere in shape.
The Leptospiraceae family was defined in 1979 to initially cover genera Leptonema as well as Leptospira. A decade ago, Levette et al. (2005) transferred Leptospira parva to genus Turneriella as Turneriella parva, contributing three genera under the family. The three genera were characterized by divergences in GC content
%, 16S rRNA gene sequences and DNA-DNA relatedness (Adler, 2015). GC content%
within members of Leptospiraceae ranged from 35 to 54 mol%. Leptospira demonstrated the lowest ratio of 35-41 mol% (Ko et al., 2009). Leptonema has a GC content% of 54 mol%, while Turneriella has the largest ratio of 53.6% mol%.
The type genus is defined as Leptospira Noguchi in the honor of Noguchi (1917) who proposed the genus name after studying the pathogen isolates from USA,
12
Japan and Europe. The type species is L. interrogans (Stimson, 1907) Wenyon 1926.
Since 1980, the type strain has been designated as serovar icterohaemorrhagiae RGAT strain (ATCC 43642) as per enlisted in Approved Lists of Bacterial Names (Skerman et al., 1980).
2.2.2 Classification of Leptospira Species And Subspecies
Three distinct classifications were used for Leptospira species, namely serology, genetic and phylogeny classification. While these classifications methods have little relatedness between each other, they present advantages and disadvantages that appropriately suit the aim of a particular study.
Since 1914, rapid isolation of the bacteria happened throughout different location of the world. Following proposal of “Leptospira” as the genus name, a number of species names have been assigned based on serological typing, such as the outcome of cross-agglutinin absorption test and difference in antigenicity of the bacteria.
Examples of Leptospira species defined by the serological typings are L. canicola, L.
hebdomadis, L. icterohaemorrhagiae and L. biflexa (Robinson, 1948). Considering inappropriate assignation of species name based on serological classification, Wolff and Broom (1954) proposed to use “serovar” for naming of serologically distinct strains. Following that, the bacteria strains were divided into two Leptospira species, namely L. icterohaemorrhagiae and L. biflexa, comprising all pathogenic and saprophytic strains, respectively. The name for pathogenic strain was then amended to L. interrogans (Wolff and Turner, 1963). To date, there are more than 250 serovars have been identified for Leptospira spp., which were grouped into 24 serogroups (Levett, 2001). Classification based on Leptospira serovars is widely used in
13
epidemiological and clinical setting. Yet, it has no relevance to the bacteria taxonomy.
In Malaysia, the three most common pathogenic Leptospira serovars are L. interrogans serovar Australis, Birkini and L. borgpetersenii serovar Javanica (Fairuz Amran, personal communication).
Methods above classifies pathogenic and non-pathogenic Leptospira into two species, namely L. interrogans and L. biflexa, respectively. With advance in genomic study, the members in the two Leptospira species were found to have low DNA homology, suggesting there are more species that laid within L. interrogans and L.
biflexa categorized using the above methods (Haapala et al., 1969). Thus, DNA-DNA hybridization technique has been widely used in many studies to characterize species within genus Leptospira. A total of 21 species have been identified to date. With the advance in molecular and analytical technique, several methods which are faster and more reliable have been prompted for species identification. For instance, MALDI- TOF has been adopted to identify Leptospira species in a recent study (Rettinger et al., 2012). Multilocus sequence typing has also been demonstrated to be able to identify the species similar to that of DNA-DNA hybridization (Boonsilp et al., 2013). It is expected that next generation sequencing will be the future trend for species identification.
By analyzing 16S rRNA and housekeeping genes such as gyrB and rrs, the Leptospira species mentioned above can be further categorized in a phylogenetic tree based on its pathogenicity (Picardeau, 2017; Morey et al., 2006; Slack et al., 2006).
As illustrated in Figure 2.3, a phylogenetic tree can be constructed into three major clades, following pathogenic, intermediate and saprophytic characteristic of the bacteria. The pathogenic Leptospira comprised species that have been characterized
14
Figure 2.3 Phylogenetic analysis of Leptospira spp. 16S rRNA and classification of Leptospira based on its pathogenicity. Reprinted from Picardeau (2017) with permission from Springer Nature.
15
to cause human and animal leptospirosis. On the contrary, species in saprophytic clade are free-living and have never demonstrated evidences in infecting a host. The intermediate Leptospira may occasionally cause leptospirosis with mild symptoms in human and animal host.
2.3 Animal Reservoir for Pathogenic Leptospira spp.
Leptospirosis has been claimed as the most common zoonotic disease. All mammals, including bats and pinnipeds, can virtually be hosts for the pathogenic bacteria (Picardeau, 2017). Unlike human who is an accidental host, leptospirosis in animal is either in asymptomatic, acute, chronic or acute-to-chronic stage with persistent bacteria carriage and excretion into the environment for a duration varies between species. As a consequence, this amplifies the infection within the ecosystem and engenders leptospirosis risk to human and animal surrounding.
Rodent is well known to be the main reservoir for Leptospira spp. and plays an important role in transmitting the disease to human and animals. As a natural reservoir, rat did not show any symptom upon infection. Although systemic infection occurs in the early stage, the bacteria is rapidly clear from blood and most organs of rats (Athanazio et al., 2008). The rat acts as a chronic carrier because the bacteria remain colonizes proximal tubules of kidney and being continuously shed into environment via urine. With bacterial load as high as 107/mL, an area may be seriously contaminated following repeating urination as the virulent leptospire is highly viable in surface water, stream, river or moist soil for weeks to months (Monahan et al., 2008).
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Domestic animals likewise impose leptospirosis risk to human as well as surrounding environment. However, these domestic animals show different degree of symptoms upon infected. Dogs demonstrate clinical presentation most resemble to human leptospirosis. Canine leptospirosis may lead to life-threatening with febrile illness symptoms in addition to vascular, liver and kidney damage (Pijnacker et al., 2016). Stray dogs transmit pathogenic Leptospira spp. from wild and natural environment close proximity to human; domestic dogs put human at the risk of contamination by shedding the bacteria in household environment (Hua et al., 2016;
Gay et al., 2014).
Staying in the same farm, livestock acquires the disease from infected herd mates and rodents that shed urine into the soil and water (Subharat et al., 2011). Since basic necessity needs (food, water, refuge) are available ad libitum in farm, it becomes a habitat for wildlife reservoirs to stay close to livestock (de Oca et al., 2017). The factors above expose workers at livestock farm to occupational-acquired leptospirosis.
Besides as a health threat to the workers, leptospirosis is associated with economic impact in livestock sector particularly with goats, sheep, pigs and cattle. In general, the infected livestock may suffer from reproduction disorders (Rizzo et al., 2017;
Ramos et al., 2006). Meanwhile, recurrent uveitis, as a result of autoimmune response between ocular tissue and leptospiral membrane proteins, is a hallmark for equine leptospirosis (Verma et al., 2013). Even though the disease is of veterinary significance, Garba et al. (2017) claimed a lack in livestock leptospirosis studies. In fact, inter-species transmission of the disease between sheep and cattle has been demonstrated in a herd two decades ago, suggesting presence of leptospirosis among livestock in Malaysia (Bahaman, 1991).
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Despite similarity in genetic, Leptospira serovars show characteristic preferences to specific animal reservoir. Icterohaemorrhagiae and Ballum serogroup are usually predominated in rats and mice, respectively (Bharti et al., 2003). Although not absolute, serovar Canicola in dogs, Harjo in cattle Pomona in pigs as well as Bratislava in horses have been primarily demonstrated (Schuller et al., 2015; Gamage et al., 2014; Andre-Fontaine, 2006; Grooms, 2006). Ellis (2010) reported that dog is the only maintenance host for serovar Canicola. Molecular background which contributes a serovar to host specificity is unknown. Frequently, these serovars do not cause severe manifestation in their highly-adapted reservoir hosts (Bharti et al., 2003).
These serovars, on the other hand, may cause severe clinical outcome to other incidental hosts.
2.4 Anatomy of Leptospira
The genus name, Leptospira, derives from Greek leptos (thin) and Latin spira (coiled).
The bacteria is a thin spirochete with approximate diameter of 0.15 µm and length of 6-20 µm. The organism is easily distinguishable from other bacteria due to their distinctive morphology of thin, right-handed helix coil and is highly motile.
Frequently, at least one end of the bacteria bends into hook which resemble a question- mark, thus contributing to the species name interrogans (“interrogate”, ask question).
Morphology of Leptospira spp. is shown in Figure 2.4.
2.4.1 Lipopolysaccharide
As illustrated in Figure 2.5A, Leptospira spp. has a Gram negative-like cell wall. The outer membrane consists of many surface exposed outer membrane (OM) proteins,
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A B
C
Figure 2.4 Morphology of Leptospira interrogans. (A) Demonstration of characteristic hook end of the bacteria. (B) Visualization wave body shape of the bacteria by scanning electron microscopy at 3000x. (C) Measurement of the bacterial cell and relative length of its endoflagellum to the cell length. (D) Schematic diagram of the bacterial cell wall. Reprinted from Bharti et al. (2003) and Picardeau (2017) with permission from Elsevier and Nature Publishing Group, respectively.
D
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B
Figure 2.5 Schematic diagram of Leptospira spp. cell wall. (A) Illustration of the bacterial cell wall architecture. It consists of outer and inner membrane, spaced by periplasm with contains endoflagellum. Lipopolysaccharide, lipoprotein, outer- and transmembrane proteins are abundantly located at the outer membrane, while several transport systems are located within inner membrane layer. (B) Schematic structure of lipopolysaccharide. It consists of repetitive O-antigen and Lipid A, linked by a core oligosaccharide region. Reprinted from Raja and Natarajaseenivasan (2015) and Erridge et al. (2002) with permission from Elsevier.
Outer membrane
Inner membrane Periplasm
Polysaccharide
