COMPARATIVE USEFULNESS OF PCR IN THE DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN DIFFERENT CLINICAL SAMPLES

Abstract

Tuberculosis (TB) remains the major cause of infectious disease in the world. About 8 million of the world's population develops the active disease annually and the problem is escalating each year with the increasing incidence of HIV. Inadequate diagnosis and resistance to current chemotherapy are also major factors in the spread of the disease.

Standard diagnosis of TB is by way of a positive Acid Fast Stain for bacillus, positive radiography and culture of the mycobacteria. Acid Fast staining for bacillus from sputum smears is fast but insensitive. Radiography indicates a general condition but is not diagnostic. Culture of the TB organism although diagnostic requires at least 3 to 6 weeks before results are available due to the slow growing characteristic of mycobacteria. New methods of diagnosis through BACTEC, serological tests and antigen detection systems (Probes) have been developed in recent years but lack sensitivity, specificity or have been too complicated and expensive for routine use. The diagnosis is especially difficult in cases where only a few mycobacteria are present.

In the past few years, the use of Polymerase Chain Reaction (PCR) for the amplification of DNA material has appeared promising in terms of direct applicability in clinical practice. Since then, various PCR assays have been developed for the rapid identification of Mycobacteria. However, they are mainly done on the pulmonary specimens such as sputum and there are few studies that tested other non-pulmonary specimens. The applicability of PCR in different clinical specimens as a feasible alternative to smear and culture remains to be established.

In this thesis, the following issues were addressed. In section 1, a general introduction and literature review on the disease tuberculosis, the PCR technique and the literature review on the applications of PCR assays in the detection of Mycobacterium tuberculosis was given. Methods used in this study were also addressed in this section. In section 2, the clinical usefulness of PCR in the detection of Mycobacterium tuberculosis in a wide spectrum of clinical specimens was addressed. The aim was to develop a rapid and simple diagnostic test that was applicable in a clinical laboratory. Details on studies and optimizations were given in this section. A short note on the laboratory protocol established and its practical use in the hospital was also given. In the last section, the usefulness of a competitive PCR diagnostic assay in defining bacterial load in an assortment of clinical specimens and in serial samples of patient undergoing anti-tuberculous treatment were assessed. Finally, in the concluding chapter, the achievement attained by the study in this thesis, its applications and limitations were addressed.