Immune markers and protective mechanisms in latent and active tuberculosis

1 1-2 Immune responses in latent tuberculosis infection LTBI .... 46 3-4.3 IFN-γ responses to PPD correlated with mycobacterium inhibition in ENR, but inversely correlated in LTBI and TB

IMMUE MARKERS AD PROTECTIVE MECHAISMS I

LATET AD ACTIVE TUBERCULOSIS

JOAE KAG SU LI

(B Sci (Hons.), US)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE

2008

ACKOWLEDGEMETS

I express my deepest gratitude to Dr Seah Geok Teng for her constant guidance, invaluable advice and unfailing patience, not forgetting the many sacrifices she had made for the past 6 years I spent under her supervision

I thank Mrs Thong for her help in acquisition of laboratory reagents and guidance in using of the flow cytometer

Special thanks to all present and past lab colleagues, particularly Eunice, Chai Lian, Irene and Baihui in processing of patient’s samples, Jamie for generating eGFP-BCG and pMV261-BCG and anyone who have supported and helped me get through this long journey

I thank Prof P Andersen and M Doherty (Statens Serum Institute, Denmark) for provision of some of the ESAT-6/CFP-10peptides, mycobacterium sonicate and PPD

I would also like to thank A/Prof YT Wang, Dr C Chee, Dr A Cherian and nursing staff (TBCU), Dr TH Lee and Dr KC Lee (Clifford Dispensary) for their help in patient recruitment and phlebotomy, as well as patients and healthy volunteers who participated in this study

Special thanks also to Fu Ling and Fui Leng (Biopolis Resource Centre) for their kind assistance and guidance in using the Cellomics ArrayScan

To my parents, sister and William, I deeply appreciate their support, patience and sacrifices made in one way or another so that I could fulfil my ambitions and dreams

Lastly, I thank everyone else who have provided me with assistance or invaluable advice in one way or another during the course of my graduate studies I could not have made it without everyone’s help!

Oral presentation:

The 5th Combined Scientific Meeting (Singapore Society for Biochemistry and Molecular Biology, Singapore Society for Microbiology and Biotechnology, Biomedical Research & Experimental Therapeutics Society of Singapore) incorporating the 4th Graduate Students’-Society – Faculty of Medicine Scientific Meeting, Singapore; 12-14 May 2004: Joanne SL Kang and GT Seah Mycobacterium-specific IFN-γ production is protective in healthy PPD+ TB contacts but not in those with latent TB infection

Poster presentation:

Novartis Institute for Tropical Diseases Inaugural Symposium on Dengue Fever and Tuberculosis, Singapore; 22-23 January 2003: JSL Kang, A Cherian, CBE Chee, TM Doherty, P Andersen, GT Seah Mycobacterial antigen recognition and immune protection against tuberculosis

TB VACCINES FOR THE WORLD - TBV 2003, Montreal, Canada; 17-19 September 2003: JSL Kang, TM Doherty, GT Seah Host immunity in tuberculosis infection

Keystone symposium: Innate immunity to pathogens, Colorado, USA; 8-13 January 2005: Joanne SL Kang and GT Seah Mycobactericidal activity of macrophages is associated with distinct T cell cytokine expression profiles in human tuberculosis

TABLE OF COTETS

Acknowledgements i

Table of Contents v Summary ix List of Tables x

List of Figures xi

List of Abbreviations xv

CHAPTER 1 PROJECT OVERVIEW AND AIMS 1

1-1 Immune responses in tuberculosis patients 1

1-2 Immune responses in latent tuberculosis infection (LTBI) 2

1-3 Research aims and project design 2

CHAPTER 2 LITERATURE REVIEW 5

2-1 Bacteriology of tuberculosis 5

2-2 Epidemiology of tuberculosis 6

2-2.1 Global burden of TB 6

2-2.2 Tuberculosis in Singapore 6

2-3 Impact of the HIV/ AIDS pandemic 8

2-4 Transmission of tuberculosis 9

2-5 Pathology of tuberculosis 9

2-6 Immunology of tuberculosis 11

2-6.1 Innate immunity 11

2-6.2 Macrophages in control of TB 12

2-6.3 Humoral immunity 13

2-6.4 Cell-mediated immunity (CMI) and T cells 14

2-6.5 Cytokines in TB 18

2-7 Diagnosis and management of tuberculosis 21

2-7.1 Diagnosis of active TB 21

2-7.2 Diagnosis of LTBI 23

2-8 Immune correlates of TB protection 25

2-9 Immune profiles in human LTBI 27

CHAPTER 3 STRONG PPD RESPONSES ASSOCIATED WITH POOR INHIBITION OF MYCOBACTERIUM GROWTH IN LATENT TUBERCULOSIS 30

3-1 Abstract 30

3-2 Introduction 32

3-3 Materials and methods 35

3-3.1 Study subjects 35

3-3.2 Antigens 37

3-3.3 PBMC stimulation and IFN-γ quantitation 38

3-3.4 Mycobacterium inhibition assay 39

3-3.5 Statistics 40

3-4 Results 41

3-4.1 PPD, ESAT-6/CFP-10 and TST responses in different clinical groups 41

3-4.2 Strong TST responders in ENR contacts show better mycobacterium inhibition than ER/LTBI contacts 46

3-4.3 IFN-γ responses to PPD correlated with mycobacterium inhibition in ENR, but inversely correlated in LTBI and TB groups 48

3-5 Discussion 53

CHAPTER 4 REGULATORY FACTORS CORRELATE WITH REACTIVITY TO MYCOBACTERIUM ANTIGENS IN ACTIVE TUBERCULOSIS 58

4-1 Abstract 58

4-2 Introduction 60

4-3 Materials and methods 62

4-3.1 Study subjects 62

4-3.2 Generation of cRNA standards 63

4-3.3 Measuring cytokine mRNA expression 65

4-3.4 Mycobacterium inhibition assay 67

4-3.5 Statistics 67

4-4 Results 68

4-4.1 LTBI TB contacts but not community controls have different cytokine profiles from ENR subjects 68 4-4.2 TB infected subjects have higher basal IFN-γ and IL-4 mRNA expression than ENR subjects 68 4-4.3 IFN-γ, TNF-α expression positively correlate with TGF-β expression in LTBI but negative correlation seen in TB 72

4-4.4 FoxP3, IL-10 and TGF-β expression are correlated in infected groups 74

4-4.5 In TB group, PPD-specific IFN-γ response associated with low TNF-α but high IL-4 and TGF-β 76 4-4.6 ESAT-6/CFP-10-specific IFN-γ response correlated with IL-4, FoxP3, TGF-β 79

4-5 Discussion 81

CHAPTER 5 DIFFERENTIAL RESPONSIVENESS TO EARLY SECRETED ANTIGENIC TARGET IN TUBERCULOSIS PATIENTS 89

5-1 Abstract 89

5-2 Introduction 90

5-3 Materials and methods 93

5-3.1 Study subjects 93

5-3.2 Quantitation of cytokine mRNA expression 93

5-3.3 Mycobacterium inhibition assay 93

5-3.4 Statistics 94

5-4 Results 95

5-4.1 ENR TB patients have higher expression of IFN-γ and TNF-α but ER TB patients have higher expression levels of regulatory biomarkers 95

5-4.2 ER TB patients have higher expression levels of suppressive factors 97

5-4.3 ER TB patients have poorer mycobactericidal activity 100

5-5 Discussion 101

CHAPTER 6 A NOVEL HIGH THROUGHPUT METHOD FOR PERFORMING THE MYCOBACTERIUM INHIBITION ASSAY TO EVALUATE HUMAN ANTI-MYCOBACTERIUM IMMUNITY 104

6-1 Abstract 104

6-2 Introduction 105

6-3 Materials and methods 108

6-3.1 Human donors 108

6-3.2 Bacteria used 108

6-3.3 Lymphocyte stimulation 109

6-3.4 BCG infection assay 110

6-3.5 BCG viability assay 110

6-3.6 ArrayScan : setting detection criteria 111

6-3.7 Computation of parameters and statistical analysis 112

6-4 Results 114

6-4.1 Fluorescence intensity of BCG with single copy versus episomal multi-copy vector 114

6-4.2 Image capture and analysis 114

6-4.3 Optimisation of lysis procedure for BCG viability assay 116

6-4.4 Correlation between fluorescence intensity and spot count 118

6-4.5 BCG-inhibition activity : dependence on lymphocytes and PPD stimulation 119

6-4.6 Correlation between parameters 122

6-4.7 Mycobacterium inhibition in latent infection versus non-infected subjects 123

6-4.8 Strong tuberculin reactivity associated with better mycobacterium inhibition 123

6-5 Discussion 127

CHAPTER 7 HOST IMMUNE PROFILES ARE ASSOCIATED WITH MYCOBACTERICIDAL ACTIVITY IN LATENT TUBERCULOSIS 134

7-1 Abstract 134

7-2 Introduction 136

7-3 Materials and methods 138

7-3.1 Study subjects 138

7-3.2 BCG inhibition and viability assays 139

7-3.3 Flow cytometry 141

7-3.4 Statistical analysis 141

7-4 Results 142

7-4.1 Significantly different cytokine profiles between polarised groups 142

7-4.2 Effect of cytokine neutralisation on mycobacterium inhibitory activity 143

145

7-4.3 Balanced cytokine profile in ESAT-responders associated with best mycobacterium inhibition 145

7-4.4 Cell surface expression of T regulatory cell markers 148

7-5 Discussion 150

CHAPTER 8 CONCLUSIONS AND FUTURE APPLICATIONS 155

8-1 Summary of key findings 155

8-2 Future work 156

8-2.1 Investigating if in vitro mycobacterium inhibition and cytokine profiles correlate with long-term protection against TB infection or reactivation 156

8-2.2 Investigating the link between environmental mycobacterium sensitisation and immune protection 157

8-2.3 Factors influencing mycobacterium inhibition 158

CHAPTER 9 REFERENCES 159

CHAPTER 10 APPENDIX 183

10-1 Middlebrook 7H9 broth (1 L) 183

10-2 Middlebrook 7H10 Agar (1 L) 183

10-3 Preparation of reagents for in vitro transcription 184

10-3.1 TE Buffer 184

10-3.2 TE-saturated phenol:chloroform: isoamyl alcohol (25:24:1) 184

10-4 Propidium iodide staining buffer 185

10-5 ESAT-6/CFP-10 peptide sequences used for stimulation 186

10-6 Chapters 3-5 patients’ demographic profiles 188

10-7 Chapters 6 and 7 subjects’ immune profiles 189

10-8 Primer sequences and PCR conditions 190

SUMMARY

There is a lack of reliable immune correlates of protection in tuberculosis (TB) This study sought to identify differences in the immune profiles of patients with active or treated TB disease, healthy TB contacts with latent tuberculosis infection (LTBI) and uninfected individuals with strong memory responses to mycobacterium antigens The work was based on analysing a combination of clinical profiles, cytokine mRNA profiles, regulatory factors, specific responses to mycobacterium

antigens and in vitro mycobactericidal activity Tuberculin reactivity, and in vitro

peripheral blood mononuclear cell responses to purified protein derivative (PPD), were both correlated with mycobactericidal activity only in clinically healthy persons without LTBI These people had better mycobacterium killing activity than those with

LTBI In TB patients, strong reactivity to Mycobacterium tuberculosis-specific

antigens ESAT-6/CFP-10 was associated with relatively lower protective and higher

T regulatory cytokine activity, consistent with their poor mycobactericidal activity The LTBI groups had a better balance of pro- and anti-inflammatory cytokines A novel high throughput intracellular mycobacterium inhibition assay technique was developed Using this assay to study TB-unexposed subjects with LTBI, the ones with

a balanced profile of pro-inflammatory and regulatory cytokines were found to be more protected than those with a skewed profile of either predominantly pro- or anti-inflammatory cytokines This study has identified biomarker profiles which characterise those with protective mycobactericidal activity, in both clinically healthy people and those with TB This is relevant to risk stratification of TB contacts and patients, and also in identifying protective responses in human TB vaccine trials

LIST OF TABLES

Table 3-1 Response rates to PPD- and ESAT-based assays in 41

different clinical groups

Table 5-1 Lack of association of clinical disease factors with 99

ESAT-response (ER or ENR) in TB patients (n = 48)

subjects

LIST OF FIGURES

Figure 3-1 Distribution of IFN-γ responses to purified protein 43

derivative (PPD) and ESAT-6/ CFP-10 in different clinical groups

Figure 3-2 Tuberculin skin test readings and correlations 45

with in vitro mycobacterium antigen-stimulated

responses in TB Contacts

inhibition in TB Contacts

Figure 3-4 Mycobacterium inhibition by 3H-uridine uptake 48

in different clinical groups

inhibition in different clinical groups

inhibition in different sub-groups of TB Contacts

Figure 3-7 Regression lines showing opposite correlation 52

trends between mycobacterium inhibition and PPD-specific IFN-γ responses in infected and uninfected groups

ESAT-responders and ESAT-nonresponders

Figure 4-2 Cytokine expression in TB patients and clinically 71

healthy subjects

(A) TNF-α or (B) TGF-β in TB patients, latent tuberculosis infection (LTBI) and ESAT-nonresponder (ENR) groups

Figure 4-4 Correlations between expression of TGF-β 73

and TNF-α in TB, LTBI and ENR groups

associated with regulatory T cells (FoxP3, TGF-β and IL-10) in TB, LTBI and ENR groups

Figure 4-6 Correlations between magnitude of PPD-specific 77

IFN-γ response and expression of TNF-α in TB, LTBI and ENR groups

Figure 4-7 Correlations between magnitude of PPD-specific 78

IFN-γ response and expression of factors associated with regulatory T cells in TB, LTBI and ENR groups

Figure 4-8 Correlations between magnitude of ESAT-6/CPF-10- 80

specific IFN-γ response and expression of factors associated with regulatory T cells in TB and LTBI groups

expression in different clinical groups (pro-inflammatory cytokines)

expression in different clinical groups (regulatory factors)

ENR TB patients and ENR Community controls

Figure 5-4 Mycobacterium inhibition by 3H-uridine uptake 100

after stimulation with PPD

Figure 6-3 Images captured by Cellomics ArrayScan in 115

BCG infection assay

Figure 6-5 Relationship between fluorescence intensity and 118

spot count

BCG-macrophage infection ratios

inhibition

Figure 6-8 Correlations between the three different readouts 122

of the BCG infection and viability assays

latent tuberculosis infection (ER) and non-infected (ENR) subjects

in subjects with different tuberculin skin test (TST) reactivity

infected macrophage and TST size

selected groups

Figure 7-3 Effects of cytokine neutralisation in total subjects 145

expressing ‘high’ levels of suppressive cytokines

cytokine polarised groups

Figure 7-5 Range of percentage viable BCG in cytokine 147

polarised groups

in cytokine polarised groups

(B) CD4+LAG3+ Treg cells by flow cytometry after PPD stimulation

LIST OF ABBREVIATIOS

cell-stimulation, e.g PPD+ means positive PPD-responder)

Mtb Mycobacterium tuberculosis

CHAPTER 1 PROJECT OVERVIEW AND AIMS

1-1 Immune responses in tuberculosis patients

Tuberculosis (TB), caused by the bacterium, Mycobacterium tuberculosis

(Mtb), is a major cause of morbidity and mortality worldwide Cell-mediated immunity is the cornerstone of host defence in TB The importance of interferon-gamma (IFN-γ) for protection is known from murine and human studies (Flynn et al., 1993; Ottenhoff et al., 1998) However, IFN-γ alone is insufficient to control infection (Flynn 1999) and strong Mtb-specific IFN-γ responses are not always correlated with protection (Elias et al., 2005) Other cytokines such as interleukin-4 (IL-4) have been associated with TB disease (Seah et al., 2000; van Crevel et al., 2000; Wassie et al., 2008) T regulatory cells (Tregs) and related cytokines such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β) have also been associated with immunosuppression in TB patients (Boussiotis et al., 2000; Guyot-Revol et al., 2006; Kursar et al., 2007; Roberts et al., 2007)

However, we still lack the full picture on the human immunological profile which predicts protection or susceptibility to TB, particularly in clinically healthy people This is partly because most studies on immune correlates of protection in such people do not take into account the heterogeneity in their prior immune exposure to mycobacteria, both pathogenic and non-pathogenic Moreover, there is difficulty in clinically defining a ‘protected’ individual, as exposure does not always lead to disease, yet it is unclear if latent infection represent a state of good protective immunity There is also a lack of a gold standard for diagnosis of latent TB infection (LTBI)

1-2 Immune responses in latent tuberculosis infection (LTBI)

Approximately 2 billion people are believed to latently infected with Mtb globally, yet little is known about host immune responses in LTBI Despite its lack of specificity, the century-old tuberculin skin test (TST), based on the crude mixture of antigens called purified protein derivative (PPD), was previously the mainstay of LTBI diagnosis Significant recent advances in mycobacterial genomics have resulted

in new diagnostic tests for LTBI detection by assaying ex vivo lymphocyte IFN-γ

responses against Mtb-specific antigens, 6kDa early-secreted antigenic target (ESAT-6) and culture filtrate protein 10 (CFP-10) However, discordant results have been noted between such IFN-γ based assays and the TST (Pai et al., 2004a), and reasons behind the discordance are not clear Two potential contributors to this phenomenon are prior host sensitisation to the widely used TB vaccine, bacille

Calmette-Guérin (BCG), and to environmental Mycobacterium species ubiquitously

found in soil and water In many TB endemic areas, clinically healthy people have varying combinations of immune exposure to BCG, Mtb and environmental mycobacteria (Black et al., 2001a), accounting for their heterogeneity in susceptibility

1-3 Research aims and project design

This project was broadly designed to understand the differences in the immunological profile of healthy persons with LTBI, and LTBI-negative people with various levels of exposure to mycobacterium antigens Additionally, it was set up to assess the extent to which various immune correlates of protection currently utilised

in this field, reflect the anti-mycobacterium immunity in clinically healthy people

living in a TB endemic area These research questions are relevant, not only to understanding the immunology of host-pathogen interactions in this disease, but also clinically for risk stratification of TB contacts and in assessing vaccine efficacy in clinical trials

The project was conducted in Singapore, where BCG is widely given at birth and there is a moderate incidence of TB Two separate cohorts of adult subjects were studied The first comprised of active and treated TB patients, TB Contacts and unexposed Community controls (Chapters 3-5) The second study cohort included only clinically healthy subjects with no defined TB exposure, but with widely varying tuberculin responses (Chapters 6-7)

Based on the first cohort, the following aims were addressed:

1 Determining rate and magnitude of responses to the TST as well as ESAT-6- and

PPD-based peripheral blood IFN-γ assays in different clinical groups, and the extent of correlation or discordance between these assays

2 Assessment of whether the magnitude of the in vitro mycobacterium inhibition

response is linked to LTBI, or the clinical status of the subjects, and the level of responses to mycobacterium antigens based on TST and in vitro IFN-γ assays

3 Investigating differences in cytokine profiles (based on unstimulated peripheral blood mononuclear cell mRNA expression) between clinical groups, and correlations between the expression of different cytokines within each group This was in order to understand how the balance of pro-inflammatory and Treg cytokines is associated with TB disease and LTBI, and to establish the relevance

of ESAT-responsiveness in TB patients

Based on the second cohort, the following aims were addressed:

4 Development of a high-throughput system for assaying, in a large number of human peripheral blood samples, the viable mycobacterium count following interactions with macrophages and lymphocytes, to overcome inadequacies of established mycobacterium inhibition assays The utility of this assay was tested

in a cohort with heterogenous TST readings

5 Assessing whether healthy unexposed subjects with different cytokine profiles and LTBI status have different mycobacterium inhibition abilities, and the effects of lymphocytes and cytokine neutralisation on mycobacterium inhibition, to determine the role of these factors in the killing of intracellular mycobacteria

6 Assessment of Treg cells and expression of Treg-associated cytokines in LTBI subjects without specific TB exposure

CHAPTER 2 LITERATURE REVIEW

2-1 Bacteriology of tuberculosis

Tuberculosis (TB) is a major cause of morbidity and mortality worldwide It is

caused mainly by the bacterium, Mycobacterium tuberculosis (Mtb) Members of the

Mycobacterium genus are aerobic and ‘acid-fast’ due to the high lipid content (high

molecular weight α-alkyl, β-hydroxy fatty acids) in the cell wall Variations in 23S rRNA internal transcriber spacer sequences between members of mycobacteria (Roth et al., 1998) has allowed speciation, but the genus is, overall, remarkably conserved with a large number of shared antigens TB is infrequently caused by other

16S-subspecies of the Mycobacterium tuberculosis complex (MTC) namely,

Mycobacterium africanum, pathogenic stains of Mycobacterium bovis and Mycobacterium microti However, other nontuberculous mycobacteria (NTM),

divided into Runyon groups according to their growth rate and pigment production,

such as Mycobacterium kansaii (Group I), Mycobacterium marinum (Group I),

Mycobacterium scrofulaceum (Group II), Mycobacterium xenopi (Group III), Mycobacterium avium-intracellulare complex (MAC) (Group III) and

Mycobacterium fortuitum (Group IV) can also cause pulmonary diseases (Katoch

2004; Glassroth 2008)

Comparative genomics finds that MTC strains differ from Mycobacterium

bovis bacille Calmette-Guérin (BCG) and many NTM in a genomic region called

region of deletion 1 (RD1) (Mahairas et al., 1996; Behr et al., 1999; Gordon et al., 1999) Deletion of RD1 from all BCG strains could be responsible for its attenuation (Cole 2002), thus secreted proteins in RD1 could have a pathogenic role (Gao et al.,

2004; Tan et al., 2006; Ganguly et al., 2008) However, gene homologues of RD1 proteins are found in a few other NTM species (Andersen et al., 2000)

2-2 Epidemiology of tuberculosis

2-2.1 Global burden of TB

In 2006, TB caused 1.7 million deaths and there were 14.4 million prevalent

TB cases, of which 9.2 million were new TB cases (139 per 100,000 population) with

an increase of 0.1 million compared to 2005 (WHO 2008) Around one third of the world’s human population is latently infected (Kochi 1991; Dye et al., 1999) This amounts to about 1.7 billion people who have been infected by Mtb, but may not exhibit symptoms It has been estimated that between 2002 and 2020, approximately

1000 million people will be newly infected, over 150 million people will get sick and

36 million will die (WHO 2008)

Most developed countries have TB incidence rates below 20 per 100,000 population; rates exceeding this are considered high by the World Health Organisation (WHO) In 2008, India, China and Indonesia are the top 3 high-burden countries globally Asia (South-east Asia and Western Pacific regions) in general, accounts for 55% of global TB cases reported, with incidence rates of 100-500 per 100,000 population per year

2-2.2 Tuberculosis in Singapore

The TB incidence rate in Singapore in 1960 was 307 per 100,000 population.This declined rapidly to 50-55 cases per 100,000 between the years 1987-1998, but

has since dropped further to 34.8 cases per 100,000 in 2006 Given the rapid decline

in incidence, older local residents had experienced a period of significantly higher TB incidence in their youth – it is unsurprising, therefore that in 2007, the incidence rate

in Singaporeans aged 60 and above was 106 per 100,000 whereas for those below 20 years, the rate was only 6 per 100,000 (Ministry of Health, Singapore April-June 2007) In addition to the increased risk of latent infection in childhood, the increased

TB incidence at older age is also likely to be due to increased risk of reactivation with declining immunity

Of all reported Singapore resident cases in 2006, 1,256 were new TB cases while 137 were relapsed cases The mortality rate of TB in Singapore in 2006 was 1.7 cases per 100,000 population (61 deaths) and accounted for 0.4% of all deaths in Singapore (Ministry of Health, Singapore April-June 2007) Based on notifications,

691 (55%) were aged 50 and above, and 863 (68.7%) were males The incidence rate was the highest amongst Malays (50.4 per 100,000); 1.5 times higher than Chinese (33.0 per 100,000) and twice that of Indians (23.8 per 100,000) In 2006, the overall rate of drug resistance was 7.8%, with 5.5% cases being resistant to one drug and 2.4% to two or more drugs (Ministry of Health, Singapore April-June 2007)

Directly Observed Treatment, Short Course (DOTS) which involves ensuring patient compliance by having them take antibiotics under observation in the outpatient setting is advocated by the WHO and has been implemented in Singapore Notification of TB cases, leading to centrally managed contact tracing, administration

of preventive chemoprophylaxis, and close monitoring of completion rates for TB treatment are additional measures taken under the Singapore TB Elimination Programme (STEP) to improve disease control Vaccination of newborn infants with

Mycobacterium bovis bacille Calmette-Guérin (BCG) began in the 1950s, with a

second booster given at 12 or 16 years of age for those with weak responses to the tuberculin skin test This revaccination policy was discontinued in 2001 in line with the 1995 WHO statement suggesting there was no additional benefit There has been approximately 95% BCG immunisation coverage in the past decade

Singapore’s neighbouring countries such as Indonesia, Philippines, Vietnam, Thailand and Cambodia have been classified by WHO to be high TB-burden countries (WHO 2008) Travel to TB endemic countries, arrival of migrant workers from these countries, and reactivation of latent infection in elderly persons who lived in Singapore during the high incidence period may be factors involved in maintaining the TB incidence in Singapore

2-3 Impact of the HIV/ AIDS pandemic

The pandemic of the human immunodeficiency virus (HIV) has changed the epidemiology of TB Due to its ability to destroy the immune system, HIV has emerged as the most significant risk factor for progression to active TB disease While

a HIV-negative, PPD-positive person has a 5-10% risk of developing active TB during their lifetime, co-infection of HIV with Mtb increases this risk to 5-15% annually (Selwyn et al., 1989) Globally, 709,000 (7.7% of 9.2 million new TB cases) new HIV-positive TB cases were estimated in 2006 and much of the increase in global TB incidence is attributable to the spread of HIV in the African Region (85%) while 6% of remaining cases were from South-East Asia, mainly India (WHO 2008)

In 2006, 357 Singapore residents were diagnosed with HIV and 3.6% of total notified

TB cases (n = 1,256) had HIV co-infection (MOH 2006; Ministry of Health, Singapore Jan-March 2007)

2-4 Transmission of tuberculosis

Inhalation of aerosolised infectious particles containing Mtb results in primary infection Following the initial primary infection, about 5-10% of individuals develop progressive primary disease within 2 years but the majority of people do not develop acute disease (Comstock 1982) Such individuals are assumed to be able to mount an effective immune response capable of either eliminating the bacteria completely or limit proliferation of the bacteria, leading to a state of latent tuberculosis infection (LTBI) As host immunity declines, about 5% of latently infected persons may develop active TB disease due to endogenous reactivation of the pre-existing dormant infection

2-5 Pathology of tuberculosis

In TB, transmission depends on aerosolisation of bacteria from airways of a person with active pulmonary TB Sputum smear-positive TB patients (Toman 1979) and those with cavitary disease have increased risk of transmission (Rodrigo et al., 1997; Reichler et al., 2002) Development of pulmonary TB occurs in several stages After entry into the lungs, the bacteria are usually ingested by mature alveolar macrophages (AMs) and destroyed within acidified phagolysomes containing high levels of lysosomal and oxidative enzymes However, if the AMs are not activated, the bacteria are not killed and may multiply logarithmically within the favourable environment of these immature macrophages Subsequently, delayed-type hypersensitivity (DTH) reactions responsible for killing mycobacterium-infected macrophages cause caseous necrosis and formation of granulomas that temporarily control and retain the bacteria Such lesions are surrounded by both activated and non-

activated macrophages, as well as T cells, thus allowing interaction and induction of cell-mediated immunity (CMI) Disease progression and subsequent manifestation of clinical symptoms are dependent on the host’s ability to develop good CMI If good CMI develops, peripheral activated macrophages continue to ingest and destroy the bacteria, and the infection is arrested at a subclinical stage However, with poor CMI, mycobacteria can replicate in surrounding non-activated macrophages Thus, the lesion enlarges and eventually erodes the bronchial wall to form a cavity and the bacteria then spread to other parts of the lung or to the external environment through aerosolisation upon coughing The bacteria can be spread via blood or lymphatics to virtually any organ in the body, including the meninges, bone, joints, reproductive organs, renal system, gut and skin Lymphadenitis and pleuritis are the most common extra-pulmonary forms of TB

It has been suggested that in LTBI, the bacteria may survive in a state of replicating persistence in the lesions due to the anoxic conditions, reduced pH, and the presence of inhibitory fatty acids (Poole and Florey 1970; Hemsworth and Kochan 1978) Hence, such dormant bacteria are not sensitive to anti-TB drugs due to their low metabolism An alternative view is that latent mycobacteria are viable for many years in the tissues of clinically healthy people, with their numbers maintained in a state of dynamic equilibrium through continuous interactions with host immunity, until the latter declines leading to uncontrolled bacterial replication and disease reactivation

non-2-6 Immunology of tuberculosis

2-6.1 Innate immunity

Natural killer (NK) cells comprise 10-15% of blood mononuclear cell population (Berke 1995) and may produce cytokines for non-specific activation of macrophages during the early phase of infection (Modlin and Barnes 1995), as well as lyse Mtb-laden cells (Scott and Trinchieri 1995)

Upon entry into the host, Mtb also interacts initially with several different antigen-presenting cell (APC) types, namely dendritic cells (DCs) and AMs in the lung airways, as well as tissue-residing monocytes and macrophages This interaction between Mtb and APCs, particularly DCs, is important as production of cytokines such as interleukin-12 (IL-12) (Henderson et al., 1997; Hickman et al., 2002) during presentation of mycobacterial antigens influences T cell activation and polarisation Heat shock protein 70 (HSP-70) produced by Mtb, binds to CD40, an important co-stimulatory molecule present on DCs and macrophages for induction of IL-12 as well

as chemokines (Wang et al., 2002) which promote granulocyte, macrophage and lymphocyte infiltration into the site of TB infection in humans Toll-like receptors (TLRs) on DCs and macrophages are receptors for several mycobacterium components (Quesniaux et al., 2004) The 19 kDa, 38 kDa lipoproteins and lipomannan (LM, a cell-wall lipoglycan) of Mtb induce IL-12 via stimulation of TLR-2 pathway (Hertz et al., 2001; Dao et al., 2004) TLR-4 signalling is protective

in murine defense against Mtb (Branger et al., 2004) and could prevent excessive chronic inflammation (Fremond et al., 2003) Another cell wall component of Mtb, lipoarabinomannan (LAM) does not induce pro-inflammatory cytokines, but induces IL-10 instead, via binding of a separate molecule, dendritic cell-specific ICAM-3

grabbing non-intergrin (DC-SIGN) (van Kooyk and Geijtenbeek 2003) DC-SIGN is a major receptor for Mtb entry into DCs (Tailleux et al., 2003) Pathogenic bacteria can even exploit TLRs or DC-SIGN to escape or suppress host immune responses (Geijtenbeek et al., 2003; Netea et al., 2004; Herrmann and Lagrange 2005)

2-6.2 Macrophages in control of TB

In lung alveoli, phagocytosis of Mtb by AMs occurs and the bacteria reside within phagosomes due to their ability to prevent phagolysosome fusion (Flynn and Chan 2003; Tufariello et al., 2003) Appropriate macrophage activation can, however, lead to some phagolysosome fusion and lysosomal enzymes (Cohn 1963), nitric oxide (NO) and reactive nitrogen intermediates (RNI) (Nicholson et al., 1996; Wang et al., 1998; Chan et al., 2001) have mycobactericidal effects Through the production of macrophage-derived cytokines such as tumour necrosis factor-alpha (TNF-α), IL-6 and IL-12 (Atkinson et al., 2000) or chemokines (Algood et al., 2003; Algood et al., 2004), other immune cells are then recruited for local inflammation and proper granuloma formation as well as presentation of mycobacterial antigens to T cells for activation and development of adaptive immunity

The activation state of macrophages is important in determining whether these cells facilitate or inhibit the growth of Mtb (Leemans et al., 2005) Resting, non-activated macrophages provide a sanctuary for intracellular replication while activation of macrophages by interferon-gamma (IFN-γ) and TNF-α increases anti-mycobacterial activity (Denis 1991; Bonecini-Almeida et al., 1998) Alternative activation of macrophages by IL-4 or IL-13, instead of classical activation by IFN-γ, during Mtb infection, leads to reduced intracellular nitrosative stress but increased

iron availability, thus supporting intracellular persistence of the bacteria (Kahnert et al., 2006).

Macrophage apoptosis may also contribute to containment of intracellular bacteria Virulent Mtb strains can prevent macrophage apoptosis (Balcewicz-Sablinska et al., 1998) while attenuated Mtb strains induce apoptosis (Keane et al., 2000), suggesting the existence of mycobacterial virulence determinants that modulate macrophage apoptosis Furthermore, macrophage apoptosis has also been associated with increased cross-priming and CD8+ T cell activation for enhanced protection against TB (Winau et al., 2004; Winau et al., 2005; Winau et al., 2006) Apoptosis of bystander uninfected macrophages in close proximity to mycobacterium-infected macrophages may limit the spread of Mtb by eliminating the bacteria’s niche cells (Kelly et al., 2008)

2-6.3 Humoral immunity

Since Mtb is an obligate intracellular pathogen, serum components are believed to have a minor protective role against TB (Johnson et al., 1997)

Anti-M bovis immunoglobulin (IgG1) responses are correlated with increased

pathology in cattle (Welsh et al., 2005) However, high IgG antibody responses to mycobacterial polysaccharides such as LAM may be protective against TB in children (Costello et al., 1992; Glatman-Freedman and Casadevall 1998) There is some evidence that mycobacterium-specific antibodies could enhance both innate and cell-mediated immune responses to mycobacteria (de Valliere et al., 2005) The potential use of monoclonal antibodies for induction of protective host-defense mechanisms against Mtb infections has been discussed (Glatman-Freedman 2006)

Activated B cells may be able to contribute to TB control by increasing production of IFN-γ by natural killer (NK) cells, as well as conferring specificity to killing of mycobacterium-infected macrophages by NK cells via antibody-dependent, cell-mediated cytotoxicity (ADCC) (Yuan et al., 1994) Major histocompatibility complex (MHC) class I cross-processing and presentation of Mtb HSP by B cells has also been suggested as an alternative pathway for generation of anti-mycobacterial CD8+ T cell responses (Tobian et al., 2005) The presence of B cells in active centres

of follicle-like structures within granulomas also suggests their contribution to antigen presentation (Ulrichs et al., 2005) B lymphocyte aggregates surrounded by macrophages or T cells in murine and human tuberculous granulomas respectively could be important for interaction with different inflammatory cell types (Tsai et al., 2006)

2-6.4 Cell-mediated immunity (CMI) and T cells

In murine tuberculosis, both activated CD4+ and CD8+ T cells accumulate in lung-draining lymph nodes within 1 week after Mtb infection (Feng et al., 1999; Serbina et al., 2000) Between 2 to 4 weeks post-infection, both CD4+ and CD8+ T cells with an effector memory phenotype (CD44hiCD45loCD62L¯) migrate to the site

of infection in the lungs, interact with APCs within granulomas (Randhawa 1990; Flynn et al., 1992) and participate in the control of the bacteria (Flynn and Ernst 2000)

The granuloma, a histopathological hallmark of TB infection, may prevent dissemination of the bacteria as well as localise inflammation to the site of infection TNF-α plays a crucial role in granuloma formation and maintenance (Flynn et al.,

1995; Bean et al., 1999; Chakravarty et al., 2008) A developing human TB granuloma comprises of a necrotic centre surrounded by an inner cell layer of APCs and CD4+ but few CD8+ T cells, and a peripheral rim of lymphocyte infiltration comprising CD4+, CD8+, B cells and mycobacterium-containing APCs arranged in active follicle-like centers that resemble secondary lymphoid organs (Ulrichs et al., 2004) These secondary lymphoid follicles could be active sites where direct cross-talk and host-pathogen interactions can occur between host immune response and Mtb (Ulrichs and Kaufmann 2006)

It has been reported that structures of murine granulomas are remarkably different from those seen in human TB (Tsai et al., 2006) and hence, the use of cynomologous macaques as a model of TB is gaining popularity, due to similarities of their granulomas and disease progression to human TB (Capuano et al., 2003; Flynn

et al., 2003) In this primate model, caseous granulomas can be detected after 4 weeks post-infection, while priming of Mtb-specific T cells occurs slowly and is detected only after 4 weeks of infection (Lin et al., 2006) Activated T cells are present earlier and at higher levels at the site of infection than in the peripheral blood A higher percentage of CD4+ than CD8+ T cells is generally activated Lung biopsies from active TB patients show the presence of follicle-like centres for host-pathogen interactions within the tuberculous granuloma (Ulrichs et al., 2005) Moreover, the distribution of macrophages and lymphocytes in lung tissues of patients with tuberculoma is different from those with cavitary TB Macrophages in tuberculoma tissues are enriched in the centre of the follicle-like structures and are surrounded by lymphocytes in a well-organised manner, while activated cells of patients with cavitary TB are disseminated, which, the authors suggest, could facilitate mycobacterium spread

The importance of CD4+ T cells in protection against TB is shown by increased pathology in mice with CD4+ cells depleted (Muller et al., 1987), adoptive transfer experiments (Orme and Collins 1983; Orme and Collins 1984) and gene knock-outs (Caruso et al., 1999) Moreover, the loss of CD4+ T cells in human HIV-

TB co-infection increases susceptibility to both acute and reactivation TB (Selwyn et al., 1989) and apoptosis of CD4+ T cells contributes to T cell hyporesponsiveness observed in peripheral blood mononuclear cells (PBMCs) of patients with active pulmonary TB (Hirsch et al., 1999b) CD4+ T cells were initially thought to be primarily for production of IFN-γ, driving nitric oxide synthase 2 (NOS2) for activation of macrophages However, studies have shown that CD8+ T cells can serve

to restore decreased IFN-γ levels and macrophage NOS2 activity after CD4-depletion

in Mtb infected mice (Caruso et al., 1999; Scanga et al., 2000) This suggests that there are IFN-γ and NOS2-independent, CD4+ T cell-dependent mechanisms for TB control A recent review discussing IFN-γ independent mechanisms used by CD4+ T cells (Goldsack and Kirman 2007) has also suggested a potential protective role for IL-17 production

For many years, the role of CD8+ T cells in protection against TB had been controversial (Mogues et al., 2001) It is now known that CD8+ T cells contribute to producing IFN-γ (Feng et al., 1999; Serbina and Flynn 1999) and lysing infected macrophages via perforin and granulysin for killing of intracellular mycobacteria (Stenger et al., 1997; Stenger et al., 1998; Serbina et al., 2000) Mtb can not only be found outside the phagosome 4-5 days after infection (McDonough et al., 1993), but presentation of mycobacterial antigens by infected macrophages to CD8+ T cells can occur as early as 12 hours after infection (Serbina et al., 2000) Mycobacteria-induced permeabilisation of the phagosomal membrane, allowing escape of the bacteria into

the cytoplasm (Teitelbaum et al., 1999), may facilitate this The phagosomal membrane is equipped with MHC class I processing machinery, thus allowing loading

of mycobacterial peptides derived from phagosomal antigens onto MHC I molecules (Guermonprez et al., 2003; Houde et al., 2003) This is known as the ‘cross-presentation’ pathway ‘Cross-priming’, which involves apoptosis of infected macrophages leading to the formation of apoptotic vesicles containing mycobacterium antigens that can be taken up by DCs, is an alternative route for Class I presentation

of Mtb antigens to CD8+ T cells (Schaible et al., 2003; Winau et al., 2004; Winau et al., 2006) There is some evidence that CD8+ T cells are required for protection against TB Latently infected humans have high frequencies of mycobacteria-specific CD8+ T cells, suggesting that these cells are important in control of replication (Lalvani et al., 1998; Lewinsohn et al., 2001) In one version of the Cornell mouse model of LTBI, depletion of CD8+T cells results in reactivation of TB (van Pinxteren

et al., 2000a)

Gamma-delta (γδ) T cells play a significant protective role against TB (Munk

et al., 1990b; Izzo and North 1992; Ladel et al., 1995), as evidenced by their accumulation in BCG-vaccinated humans and PPD-reactive latently infected individuals (Tsukaguchi et al., 1995; Li et al., 1996; Hoft et al., 1998) Mtb-reactive

γδ T cells that secrete cytokines involved in granuloma formation have also been found in the peripheral blood of tuberculin-positive healthy individuals (Munk et al., 1990a) Other γδ T cell effector functions, such as production of IFN-γ, TNF-α or IL-

17, their capacity to mediate cytolysis, and reduce extracellular and intracellular Mtb growth have also been reported (Tsukaguchi et al., 1995; Boom 1999; Thoma-Uszynski et al., 2000; Dieli et al., 2001; Lockhart et al., 2006) However, another study also shows that increased numbers of lytic γδ T cells in TB contacts do not

protect these individuals from subsequent TB disease development (Ordway et al., 2005)

2-6.5 Cytokines in TB

IFN-γ and TNF-α both play central roles in the control of TB in mice and humans since the lack of functional IFN-γ or its receptor enhances TB susceptibility (Cooper et al., 1993; Flynn et al., 1993; Ottenhoff et al., 1998) while neutralisation of TNF-α increases reactivation of TB disease (Keane et al., 2001; Mohan et al., 2001) Both cytokines also work synergistically to activate macrophages and induce anti-mycobacterium effects via the production of RNI (Flesch and Kaufmann 1990) However, mycobacterium growth inhibition does not correlate with IFN-γ responses (Hoft et al., 2002) and IFN-γ may even enhance intracellular Mtb growth (Douvas et al., 1985) IFN-γ may possibly have a suppressive regulatory role preventing maintenance of Th1 memory cells (see review (Goldsack and Kirman 2007)) High levels of TNF-α are also known to lead to increased pathology and necrosis This is a major factor in host-mediated destruction of lung tissue in both humans and mice (Rook et al., 1987; Moreira et al., 1997) Moreover, the presence of both cytokines at sites of TB infection has been found to accentuate apoptosis of Mtb-responsive T cells leading to persistence of TB (Hirsch et al., 2001)

Production of IFN-γ is induced by IL-12 and IL-18 (Trinchieri and Gerosa 1996; Dinarello et al., 1998; Yoshimoto et al., 1998) and possibly IL-23 (Oppmann et al., 2000) IL-12 is produced by macrophages and DCs after phagocytosis of Mtb (Henderson et al., 1997; Ladel et al., 1997) and is composed of the 35 and 40 kDa subunits; IL-23 has a p19 subunit and shares the p40 subunit with IL-12 (Oppmann et al., 2000) The importance of the p40 subunit but not the p35 subunit for inducing

protective IFN-γ responses to Mtb is seen in a murine study using p40 and p35 deficient mice (Cooper et al., 2002) While IL-18 alone is unable to compensate for a dysfunctional IL-12 pathway (Verreck et al., 2002), IL-23 has been found to restore immunity during Mtb infection in the absence of functional IL-12p70 (Khader et al., 2005; Wozniak et al., 2006) IL-23 has variously been described as being necessary for induction of Th17 cells during Mtb infection (Khader et al., 2005) or not essential for the development of protective IL-17 secreting T cells (Wozniak et al., 2006) Induction of IL-17-secreting cells is inhibited by IFN-γ and IL-4 and the inhibition of IFN-γ enhances development of Th17 cells (Harrington et al., 2005) Some studies have shown that transforming growth factor-beta (TGF-β) is necessary to induce development of Th17 cells (Mangan et al., 2006) while others show that IL-1β and IL-6, not TGF-β, are the key components for differentiation of IL-17 producing cells (Acosta-Rodriguez et al., 2007)

The role of IL-6 in TB is still unclear due to conflicting data IL-6 produced by BCG-infected macrophages has been implicated in the suppression of T cell responses (VanHeyningen et al., 1997) and promotes intracellular mycobacterium growth in macrophages (Denis and Gregg 1990) by inhibiting bacteriostatic effects of TNF-α (Bermudez et al., 1992) However, other studies have also shown that IL-6 is required for the priming of protective IFN-γ producing Th1 cells during immunisation with a

TB subunit vaccine (Leal et al., 1999; Leal et al., 2001a) and for stimulating early IFN-γ production and control of Mtb in the early stage of infection (Saunders et al., 2000)

IL-10 and TGF-β have been implicated in the suppression of immune responses in TB patients (Rojas et al., 1999; Boussiotis et al., 2000; Bonecini-Almeida et al., 2004) Both possess macrophage deactivating properties (Ding et al.,

1990; Gong et al., 1996) and are produced by T regulatory cells (Boussiotis et al., 2000; Roberts et al., 2007) In mice, the absence of IL-10 results in increased resistance towards mycobacterial infections (Murray and Young 1999; Jacobs et al., 2000) while over-expression of IL-10 reactivates chronic TB infection with resultant increased mycobacterial load (Turner et al., 2002)

TGF-β is known to inhibit many IFN-γ mediated mechanisms (Gorelik and Flavell 2002) including inhibition of T-bet expression and thus, blocking Th1 differentiation (Gorelik et al., 2002) TGF-β is found in lung lesions of patients with pulmonary TB and excess production of TGF-β is correlated with advanced TB, suggesting a role for TGF-β in undermining anti-mycobacterium immune responses and contributing to increased pathology (Aung et al., 2000)

IL-4 is another cytokine that is associated with TB disease (as reviewed by (Rook 2007) and is predominantly produced by Th2 cells However, IL-4 has also been found to be produced by T regulatory (Treg) cells together with IL-10 and TGF-β (Zelenika et al., 2002) In a recent study, decreased IFN-γ, accompanied by increased IL-4, TGF-β and FoxP3 mRNA expression, has been found in TB patients The authors suggest that immunosuppression in active TB disease is attributable to increased Treg activity (Roberts et al., 2007) Furthermore, IL-4 can directly affect macrophage activation and function, and result in alternatively activated macrophages with diminished protective responses against intracellular Mtb (Kahnert et al., 2006),

as well as inhibit autophagy-mediated killing of intracellular mycobacteria by macrophages (Harris et al., 2007) A shift from a Th1-IFN-γ biased response, towards

a Th2-IL-4 response with increased humoral responses, during TB disease progression in cattle, has been associated with more extensive pathology (Welsh et al., 2005)

2-7 Diagnosis and management of tuberculosis

2-7.1 Diagnosis of active TB

TB infection induces clinical symptoms which typically include fever, loss of weight and persistent coughing Diagnosis usually involves physical examination, radiologic chest imaging and bacteriological diagnosis via microscopy and acid-fast staining of sputum samples, as well as culture of the bacteria Apart from traditional sputum staining by Ziehl-Neelsen or Kinyoun techniques, newer fluorochrome staining procedures using auramine O or auramine-rhodamine dyes, could be more sensitive (Zheng and Roberts 1999) However, although sputum smears are a rapid means for mycobacteria detection in clinical samples, high percentage of false negative results may still occur due to the patient’s inability to produce a good sputum sample Moreover, the detection limit for mycobacteria on smears is approximately

104-105 bacteria/ml of sputum Although the detection limit for cultures is about 100 bacteria/ml, prior decontamination of sputum specimens to inhibit growth of oral flora

is necessary for good recovery Culture is very slow, requiring 6-8 weeks of incubation for visible colony formation in some cases (Warren and Body 1995)

The BACTEC system for radiometric detection of mycobacteria growing in broth has a shorter detection time of 5-10 days and simultaneously allows drug susceptibility testing (Roberts et al., 1983), but its high cost and use of radioactivity are the drawbacks The use of an acridinium ester-labeled, single stranded DNA probe (Gen-Probe) which binds mycobacteria rRNA for rapid identification of mycobacteria

in clinical laboratories was introduced in the 1990s (Walker and Dougan 1989) and is known to have high sensitivity and specificity

Microscopic-observation drug-susceptibility (MODS) assay is a new rapid and more sensitive method for detection of TB from sputum culture It has been shown to

be useful for distinguishing between TB patients and healthy controls (Caviedes et al., 2000; Moore et al., 2004), as well as drug susceptibility testing (Moore et al., 2006; Bwanga et al., 2009) It is dependent on the following three principles: 1) Mtb grows faster in liquid broth than solid agar, 2) characteristic cord-like structures can be viewed via microscopy in liquid media, and 3) a direct association between detection

of Mtb growth and incorporation of anti-TB drugs

High levels of Mtb-specific antibodies can be found in TB patients and there has been interest in using this for diagnosis (Uma Devi et al., 2001; Raja et al., 2002; Ramalingam et al., 2002; Araujo et al., 2004; Raja et al., 2004) It could be particularly useful in diagnosis of HIV-TB coinfected individuals (Ramalingam et al., 2003; Uma Devi et al., 2003) However, a recent review assessing the accuracy of various commercially available antibody detection tests reported the inadequacy of such serodiagnostic tests for diagnosis of pulmonary tuberculosis and that none of the assays performed well enough to replace sputum smear microscopy (Steingart et al., 2007) This could be due to the use of non-specific mycobacterial antigens employed

in the tests Humans and most animals often have repeated exposure with NTM that have lesser tendency to cause disease, but nonetheless induce antibody responses, thus

leading to high number of false-positive results

The Stop TB campaign launched by WHO in 2006 aims to halve TB prevalence and death rates globally compared to the level in 1990 by the year 2015 (WHO 2008) Treatment of active TB disease following diagnosis and drug susceptibility testing consists of administration of a combination of 4 anti-TB drugs, comprising usually of isoniazid, rifampicin, ethambutol and pyrazinamide for a

minimum period of 6 months under the directly observed treatment programme Adherence to therapy is necessary to prevent acquired drug resistance Globally, WHO estimates that 424,000 new cases of multi-drug resistant (MDR)-TB strains (resistant to first line-drugs, isoniazid and rifampicin) arise yearly while 25,000 to 30,000 new cases of XDR-TB (resistant to all effective anti-TB drugs) have been reported (Ministry of Health, Singapore April-June 2007) Treating MDR-TB may be

1000 times more costly than treating standard TB

2-7.2 Diagnosis of LTBI

Because Mtb can persist within macrophages or other cells in a state of dormancy with no replication and little metabolism, an individual with LTBI appears asymptomatic and is non-infectious Hence, LTBI cannot be diagnosed with the usual tools such as chest X-ray, sputum smears or culture Although DTH responses to Mtb antigens may not directly correlate with immune protection or TB pathology (Hart et al., 1967; Fine et al., 1994), in clinical practice, the Mantoux or tuberculin skin test (TST) which is based on the detection of DTH to purified protein derivative (PPD) of Mtb, is often the basis for TB contact screening for LTBI and chemoprophylaxis to prevent disease reactivation (Jasmer et al., 2002) Limitations to the use of PPD of

Mtb in the TST include cross-reactions in subjects with prior BCG vaccination

(Menzies and Vissandjee 1992), or prior immune exposure to environmental NTM (Palmer and Long 1966) Thus, use of TST for LTBI diagnosis is particularly problematic in areas with a childhood BCG vaccination and revaccination programme, because TST induration sizes increase with number of BCG revaccinations (Chee et al., 2001) However, a study in Turkey, where TB prevalence