Project topics and materials
Get Complete Project Material File(s) Now! »
LITRATURE REVIEW
Introduction
Tuberculosis (TB) is a disease of major public health concern worldwide. The World Health Organization (WHO) estimated that 9.4 million new cases of TB occurred in 2009 (WHO, 2010a). The high incidence of TB is further compounded by the increasing problem of multidrug-resistant (MDR) Mycobacterium tuberculosis (M. tuberculosis) strains that are resistant to isoniazid (INH) and rifampicin (RIF) (WHO, 2010a). In 2008, an estimated 440 000 cases of MDR-TB were diagnosed, of which approximately 40 000 were extensively drug-resistant (XDR)-TB [ie MDR-TB with additional resistance to any fluoroquinolon (FLQ) and to at least one of the three injectable second-line drugs, kanamycin (KAN), amikacin (AMK) and/or capreomycin (CAP)] (CDC, 2006; WHO-IUATLD, 2008; WHO, 2010b). In 2005, outbreaks of XDR-TB have been reported in Russia and South Africa (WHO, 2008a). By January 2010, XDR-TB cases had been reported in 58 countries around the world (WHO, 2010b).
Treatment of MDR-TB is complex and uses second-line anti-TB drugs that are less effective, and toxic that must be administered for a longer duration than for drug-susceptible TB patients (Orenstein et al., 2009). Patients with MDR-TB have lower cure rates and higher mortality than patients with drug-susceptible TB (Orenstein etal.,2009). However, successful outcomes for MDR-TB are achievable in about two-thirds of patients (Orenstein et al., 2009). Outcomes of treatment for XDR-TB vary in different countries (Gandhi et al., 2006; Mitnick et al., 2008; Orenstein et al., 2009; Dheda et al., 2010).
With the global rise in MDR-TB strains, there is an increasing need to determine susceptibility to first and second-line anti-TB drugs accurately. The available conventional methods for mycobacterial drug susceptibility testing (DST) are based on solid media (Heifets, 1991; Heifets and Desmond, 2005; Parsons et al., 2011). The proportion method using either Lowenstein-Jensen (LJ) or agar medium is universally accepted as the ‘‘gold standard’’ although it has longer turnaround times before final results are available (Heifets, 1991; Heifets and Desmond, 2005; Parsons et al., 2011). Rapid DST methods including phenotypic and genotypic assays have been developed for first-line anti-TB drugs to shorten the time of susceptibility testing of M. tuberculosis. However, DST methods for second-line anti-TB drugs are not yet standardised and are less reproducible than methods for first-line anti-TB drugs (Shah et al., 2007). Critical drug concentrations for second-line anti-TB drugs have not been completely established for all the drugs (Shah et al., 2007). As a result, the methods and drug concentrations used for second-line anti-TB drugs vary between different laboratories (Shah et al., 2007).This highlights the urgent need for studies on accurate susceptibility testing in order to standardise methods and drug concentrations for second-line drugs.
In addition to the management of drug-resistant strains,understanding the population structure, diversity and spread of resistantM.tuberculosis strains is crucial.Molecular characterisation of drug-resistant strains is helpful to gain insight in the major circulating strains of M.tuberculosis (Mathema et al., 2006). A number of genotyping methods based on various genetic markers have been developed (Moström et al., 2002).These methods include IS6110-based restriction fragment length polymorphism (RFLP), spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. The IS6110-RFLP typing has been the most widely used and is internationally accepted as the gold standard and has become invaluable in investigations of disease transmission dynamics and outbreaks (Mathema et al., 2006). However, IS6110-RFLP typing is a laborious method that requires large amounts of DNA per isolate and has poor discriminatory power for M. tuberculosis isolates with a low copy number of IS6110 (Van Embden et al., 1993). It is important to combine various typing methods to increase the power of strain differentiation as a single genotyping method cannot define all unique isolates (Barlow et al., 2001; Cowan et al.,2005).
Classification of mycobacteria
The genus Mycobacterium is the only genus in the family Mycobacteriaceae (Barrera, 2007; Pfyffer, 2007). The genus Mycobacterium is classified into several species (Pfyffer, 2007; Euzéby, 2011). However, for the purpose of the diagnosis and treatment, Mycobacterium species can be grouped into M. tuberculosis complex and nontuberculous mycobacteria (Barrera, 2007; Pfyffer, 2007). The M. tuberculosis complex members are causative agents of human and animal TB (Pfyffer, 2007). Species in this complex include: M. tuberculosis, (the major cause of human TB), M. bovis (cattle strain), M. bovis BCG (vaccine strain), M. africanum, M. canetti, M. caprae, M. microti and M. pinnipeddii (Pfyffer, 2007; Euzéby, 2011). Nontuberculous mycobacteria include M. avium, M. kansasii, M. intracellulare and M. fortuitum (Pfyffer, 2007). Nontuberculous mycobacteria can cause pulmonary disease resembling tuberculosis, lymphadenitis and skin disease or disseminated disease (Pfyffer, 2007; Euzéby, 2011).
General characteristics of mycobacteria
Mycobacteria are aerobic, non-motile, non-sporulated rods and do not contain a capsule nor produce any toxins (Barrera, 2007; Pfyffer, 2007). All Mycobacterium species share a characteristic cell wall, thicker than in many other bacteria, which is hydrophobic, waxy and rich in mycolic acids/mycolates (Draper and Daffé, 2005; Barrera, 2007; Bhamidi, 2009). The high content of complex lipids of the cell wall prevents access of common dyes (Pfyffer, 2007; Bhamidi, 2009; Todar, 2011). Therefore, the Mycobacterium species, along with members of a related genus Nocardia, are classified as acid-fast bacteria (Barrera, 2007; Todar, 2011). Despite this, once stained, acid-fast bacteria will retain dyes when heated and treated with acidified organic compounds (Barrera, 2007; Pfyffer, 2007; Todar, 2011).Mycobacteria are widespread bacteria, typically living in water (including tap water treated with chlorine) and food sources (Barrera, 2007). However, mycobacteria such as M. tuberculosis and M. leprae are obligate pathogens; M. avium is opportunistic pathogens while other species are saprophytes (Pfyffer, 2007). Mycobacterium tuberculosis can infect several animal species, although humans are the principal hosts (Grange, 2009). Mycobacterium tuberculosis grows most successfully in tissues with high oxygen content, such as the lungs (Grange, 2009; Lawn and Zumla, 2011).
Colony morphology of mycobacteria varies among species, ranging from smooth to rough and from nonpigmented to pigmented (Pfyffer, 2007). Two types of media are used to grow M. tuberculosis namely Middlebrook medium, which is an agar-based medium and Löwenstein-Jensen (LJ) medium which is an egg-based medium (Barrera, 2007; Pfyffer, 2007). Both types of media contain inhibitors to prevent contaminants from out-growing M. tuberculosis (Barrera, 2007; Pfyffer, 2007). Mycobacterium tuberculosis colonies are small and buff coloured when grown on either LJ or Middlebrook media (Barrera, 2007; Pfyffer, 2007). The generation time of M. tuberculosis is 15 to 20 hours, which is slow compared with other bacteria [Escherichia coli (E. coli) divides every 20 minutes] (Pfyffer, 2007; Lawn and Zumla, 2011). It takes 4 to 6 weeks to get visual colonies on either type of media and M. tuberculosis tends to grow in parallel groups, producing serpentine cording (Barrera, 2007; Pfyffer, 2007).
Virulence factors of M. tuberculosis
Unlike other bacteria, M. tuberculosis does not possess the classic bacterial virulence factors, such as toxins, capsules and fimbriae (Todar, 2011). However, a number of structural and physiological properties of the M. tuberculosis have been described that contributes to mycobacterial virulence and the pathology of TB, even though the exact role in tuberculosis virulence is unclear (Todar, 2011). These include: (i) M. tuberculosis have special mechanisms for cell entry by binding directly to mannose receptors on macrophages (ii) M. tuberculosis can grow intracellularly in macrophages.Resistance to killing by macrophages is critical to the virulence of M. tuberculosis. Macrophages produce reactive oxygen species and reactive nitrogen species that have potent antimicrobial activity. Mycobacterium tuberculosis has two genes encoding superoxide dismutase proteins, sodA and sodC. SodC is a Cu, Zn superoxide dismutase responsible for only a minor portion of the superoxide dismutase activity of M. tuberculosis. However, SodC has a lipoprotein binding motif, which suggests that it may be anchored in the membrane to protect M. tuberculosis from reactive oxygen intermediates at the bacterial surface. (iii) M. tuberculosis interferes with the toxic effects of reactive oxygen intermediates produced in the process of phagocytosis (iv) antigen 85 complex: this complex is composed of a group of proteins secreted by M. tuberculosis and these proteins help in walling off the bacilli from the immune system and may facilitate tubercle formation (v) slow generation time, the immune system may not readily recognize the bacilli or may not be triggered sufficiently to eliminate the M. tuberculosis (vi) high lipid concentration in cell wall which accounts for impermeability and resistance to antimicrobial agents (vii) cord factor: the cord factor is a surface glycolipid which blocks macrophage activation by IFN-γ, induces secretion of TNFα and causes M. tuberculosis to form cords in-vitro.This is the main virulence factor which helps tuberculosis to become resistant to anti-TB drugs (Todar, 2011).
SUMMARY
LIST OF ABBREVIATIONS
LIST OF TABLES
LIST OF FIGURES
LIST OF PUBLICATIONS AND CONFERENCE CONTRIBUTIONS
CHAPTER 1: INTRODUCTION
CHAPTER 2: LITERATURE REVIEW
2.1 Introduction
2.2 Classification of mycobacteria
2.3 General characteristics of mycobacteria
2.4 Virulence factors of M. tuberculosis
2.5 Pathogenesis of M. tuberculosis
2.6 Treatment of TB
2.7 Drug-resistance in M. tuberculosis
2.8 Epidemiology of drug-resistant TB
2.9 Genetic basis of resistance against first and second line anti-TB drugs
2.9.1 Isoniazid
2.9.2 Rifampicin
2.9.3 Pyrazinamide
2.9.4 Streptomycin
2.9.5 Ethambutol
2.9.6 Kanamycin, amikacin, capreomycin and viomycin
2.9.7 Fluoroquinolones
2.9.8 Ethionamide
2.9.9 Serine analogues
2.10 Diagnosis of drug-resistant TB
2.10.1 Phenotypic susceptibility testing techniques for M. tuberculosis
2.10.1.1 Conventional solid culture-based susceptibility testing methods for M. tuberculosis
2.10.1.1 Rapid solid culture-based susceptibility testing methods for M. tuberculosis
2.10.1.2 Liquid culture-based susceptibility testing methods for M. tuberculosis
2.10.2 Molecular detection of drug-resistance in M. tuberculosis
2.10.3 Molecular epidemiology of M. tuberculosis
2.10.4 Genotyping methods used for M. tuberculosis
2.10.4.1 IS6110-RFLP genotyping of M. tuberculosis
2.10.4.2 Spoligotyping of M. tuberculosis
2.10.4.3 Mycobacterial interspersed repetitive unit-variable number tandem repeat typing of M. tuberculosis
2.10.5 Strain families of M. tuberculosis
2.10.5.1 The East African-Indian family
2.10.5.2 The Beijing family
2.10.5.3 The CAS or Delhi lineage
2.10.5.4 The Haarlem family
2.10.5.5 The LAM family
2.10.5.6 The X family
2.10.5.7 The T families
2.11 Summary
References
CHAPTER 3: COMPARISON BETWEEN THE BACTEC MGIT 960 SYSTEM AND THE AGAR PROPORTION METHOD FOR SUSCEPTIBILITY TESTING OF MULTIDRUG RESISTANT TUBERCULOSIS STRAINS IN A HIGH BURDEN SETTING OF SOUTH AFRICA
3.1 Background
3.2 Results
3.3 Discussion
3.4 Methods
3.4.1 Study design
3.4.2 Specimens
3.4.3 Routine drug susceptibility testing using BACTEC MGIT 960
3.4.4 Drug susceptibility testing using the agar proportion method
3.4.5 Analysis
Acknowledgements
Reference
CHAPTER4: EVALUATION OF THE GENOTYPE® MTBDRSL ASSAY FOR SUSCEPTIBILITY TESTING OF SECOND-LINE ANTI TUBERCULOSIS DRUGS
4.1 Introduction
4.2 Materials and Methods
4.2.1 Study setting and clinical isolates
4.2.2 Drug susceptibility testing
4.2.3 GenoType® MTBDRsl assay
4.2.4 DNA sequencing of discrepant results
4.2.5 Statistical analysis
4.3 Results
4.4 Discussion
Acknowledgements
References
CHAPTER 5: MOLECULAR CHARACTERIZATION AND SECOND-LINE ANTI-TB DRUG-RESISTANCE PATTERNS OF MULTIDRUG RESISTANT TUBERCULOSIS ISOLATES FROM THE NORTHERN REGION OF SOUTH AFRICA
4.1 Introduction
4.2 Materials and Methods
5.2.1 Study population
5.2.2 Routine culture and drug susceptibility testing
5.2.3 Drug susceptibility testing
5.2.4 Genotyping
5.2.5 Data analysis
5.3 Results
5.4 Discussion
5.5 Conclusions
5.6 Acknowledgements
5.7 References
CHAPTER 6: CONCLUDING REMARKS
Future research
References
APPENDIX A: DETAILED METHODOLOGY
APPENDIX B: DATA AND DETAILED RESULTS
