الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Tuberculosis (TB) is a progressive granulomatous infectious disease caused by M. tuberculosisand it primarily affects the lungs causing PTB.
However, lymphatic and haematogenous dissemination of MTBbacilli can result in infection of any organ especially the lymph nodes, pleura, bones, meninges and urinary tract.
The world has witnessed a significant increase in TB incidence since 1980’s and since then, TB has been considered a major public health problem not only in developing countries, but also in industrialized countries.
The emergenceof MDR and XDR forms of MTB, resulting from the misuse of the few available anti-TB drugs, has made TB treatment and prevention a permanent challenge over the course of human history.The social factors which play a role in TB transmission include poor quality of life, poor housing, overcrowding, malnutrition, smoking, alcohol abuse, lack of education and lack of awareness regarding the cause and transmission of TB.
Based on the latest WHO global TB report (2019), the world as a whole is currently not on track to reach the 2020 milestones of the “End TB Strategy”, where the global reduction in TB incidence within the last few years was less than one third of the way towards the End TB Strategy milestone of a 20% reduction between 2015 and 2020.
The same non-promising global reduction rate applies to the number of TB deaths which was only 11% reduction; a value much less than the End TB Strategy milestone of a 35% reduction by 2020.Nowadays, pipelines for new TB diagnostics, drugs, and vaccines are in a continuous progress, but in a very slow manner.
Therefore, a critical step in the control of TB is the early and accurate diagnosis of the disease in order to break the chain of its transmission and to meet the global targets for 2020–2035.By reviewing scientific literature, many attempts have been carried out to improve the performance and accuracy of several TB diagnostic tests, including smear microscopy, culture-based tests, NAA tests. Nonetheless, all of these tests suffer from a number of drawbacks which limit their widespread use.Serological antibody detection tests are considered convenient, simple to use and economical diagnostic alternative in low income countries.
However, diagnosing TB serologically is a great challenge, because of the highly variable values for test sensitivity and specificity.
On the other hand, the approach of TB diagnosis through detection of mycobacterial antigens in clinical specimens is a promising approach for the reliable diagnosis of TB.
However, research and development in the area of TB antigen detection is very limitedand needs to be accelerated.Therefore, the current study aimed to investigate the use of different potential MTB antigenic biomarkers in patient serum for TB diagnosis using a reliable diagnostic tool, in an attempt to overcome one of the main obstacles of controlling a global TB epidemic.
Atotal of six TB antigens, Rv0934, Rv0819, Rv0388c, Rv2200c, Rv1970 and Rv2068c, having high immunogenicity were selected. Five of these antigens were successfully expressed by recombinant DNA techniques and were purified in a denatured form. Threeof these five antigens were also purified in a native form.
Upon using these individual recombinant antigens in pilot trials for antibody detection in patient serum samples by indirect ELISA, the test sensitivity was unacceptably low (less than 25%).
Summary, conclusions and recommendations115Consequently, we started our trials for TB diagnosis by direct immunodetection of mycobacterial antigenic biomarkers in patient serum specimens using different techniques; sandwich ELISA, competitive ELISA and dot blot.
Specific polyclonal antibodies to the individual recombinant antigens were raised in mice and rabbits and were further used in the detection of soluble serum antigens in several pilot trials of sandwich ELISA.
Although the test design was successful, no promising results were obtained.Specific murine polyclonal antibodies were used for Rv0819 antigen detection by dot blotassayand competitive ELISA, where the former proved to be more sensitive and specific for the detection of such antigen. To our knowledge, this is the first study that demonstrates Rv0819 as a potential TB serum biomarker where it was detectable in the sera of a significant number of newly diagnosed TB patients with 100% specificity by dot blot assay.
However, the detection of Rv0934 in patient sera using the specific murine polyclonal antibodies by dot blot assay was not successful due to the presence of this antigen in the serum of BCG-vaccinated individuals causing false-positive results.
Phage display technology was used for the selection of monoclonal antibodies(ScFv) against each of the native antigens, Rv0934 and Rv0819. The selected most reactive clone for each antigen was used as a primary antibody solution for thedetection of the respective antigen in a significant number of patient sera using phage dot blot assay with high sensitivity; 91.67% in case of Rv0934 antigen detection and 87.18% in case of Rv0819 antigen detection.