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Sputum – smear microscopy
Microscopy still remains the cornerstone of TB diagnosis due to its cost-effectiveness, rapid analysis and minimal requirements for infrastructure or equipment. The first observation of the tubercle bacilli was performed by Robert Koch in 1882. Using methylene blue staining he described the “beautifully blue” aetiological agent, known today as M. tuberculosis, as being responsible for TB infection (Koch, 1982). A few weeks after this, Paul Ehrlich discovered the acid-fastness of mycobacteria, a characteristic which allows only mycobacteria stained with arylmethane dyes to retain its color after decolourization with acid alcohol solutions (Barrera, 2007). ZN staining is an adaptation of Ehrlich’s staining method and became the widely adopted technique for detection of M. tuberculosis (Titford, 2010). The staining, together with the distinct cording morphology of a select few mycobacteria allows smear microscopy to be highly specific in identifying TB positive cases. ZN-stained smears of sputum specimens examined by conventional light microscopy remains the primary tool for diagnosing pulmonary TB in disease endemic countries. It is rapid and
inexpensive to perform, with high specificity for the detection of infectious cases of TB in high prevalence areas (Steingart et al., 2006) While the technique is highly specific, its sensitivities range from anywhere between 35-70 % making it a poor diagnostic. In general, approximately 50% of culture-positive cases are detected by microscopy (Perkins, 2000, Kivihya-Ndugga et al., 2003, Steingart et al., 2006). Diagnostic sensitivity has been associated with the skill of the microscopist, with training taking at least 2 weeks or longer prior to a confident diagnoses being made (Boehme et al., 2011). Furthermore, the inadequate production of sputum in HIV positive patients have resulted in a lower proportion of smear positive TB cases detected (Carman and Patel, 2014).
Interferon gamma release assays (IGRA)
Repeated exposure of T-cells to antigens of M. tuberculosis bacteria results in the production of IFN-γ and can then be detected. IGRA assays are based on the release of IFN-γ after exposure of either whole blood or peripheral blood mononuclear cells to a M. tuberculosis specific antigen (Cattamanchi et al., 2011). The use of an M. tuberculosis-specific antigen rules out the effect of prior BCG vaccination and of non-tuberculous mycobacteria. Two blood tests are currently available based on in-vitro stimulation of T-lymphocyte cells, using antigens unique to M. tuberculosis, to release IFN-γ. One assay, the enzyme-linked immunospot (ELISpot) [T-SPOT.TB; Oxford Immunotec; Oxford, UK] estimates Tlymphocyte cells secreting IFN-γ, while the other assay uses an enzyme-linked immunosorbent assay (ELISA) to measure secreted IFN-γ concentrations [QuantiFERON-TB Gold; Cellestis; Carnegie, Australia]. Several studies confirm the higher specificity of these assays compared to the TST, due to the uniqueness of the antigens which are absent from BCG and other non-tuberculous mycobacteria and are therefore not confounded by previous exposure to the BCG vaccine and environmental mycobacteria (Kariminia et al., 2009, Katsenos et al., 2010). A drawback of these tests is their inability to distinguish active disease from latent infection, keeping the specificity for active disease low especially in high prevalence areas (Pai and Menzies, 2007). For both active TB disease and latent tuberculosis infection, evidence suggests the ELISA has similar sensitivity to the TST skin test, while the ELISpot is appears more sensitive (Kang et al., 2007, Pai et al., 2007, Pai and Menzies, 2007, Dyrhol-Riise et al., 2010, Cattamanchi et al., 2011, Pai et al., 2014).The United States of America Food and Drug Administration (FDA) has approved both QuantiFERON-Gold-In-Tube and T-Spot as in vitro diagnostic for the indirect detection of M. tuberculosis infection when used together with risk assessment, radiography, and other medical and diagnostic suggestions (Mazurek et al., 2010). Of note, is the high incidence of latent TB or sensitization of T-cells to TB antigens due to continuous exposure of individuals to TB sufferers, which affects the performance of IGRAs in TB burdened countries (Rangaka et al., 2007, Zwerling et al., 2012, Zwerling et al., 2013).
CHAPTER 1 INTRODUCTION
CHAPTER 2 LITERATURE REVIEW
1. Introduction
2. The Tubercle bacillus
3. Tuberculosis
4. Pulmonary specimens
5. Sputum collection and processing
6. Biosafety requirements of a tuberculosis laboratory
7. Processing sputum for direct sputum – smear microscopy
8. Diagnostics
9. Summary
10. References
CHAPTER 3
1. ABSTRACT
2. INTRODUCTION
3. MATERIALS AND METHODS
4. RESULTS
5. DISCUSSION
6. CONCLUSIONS
CHAPTER 4
1. ABSTRACT
2. INTRODUCTION
3. METHODS
4 RESULTS
5 DISCUSSION
6 CONCLUSION
CHAPTER 5 CONCLUDING REMARKS
