الفهرس 
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Abstract
Tuberculosis is a chronic universal disease caused by the genus of Mycobacterium tuberculosis. The disease is responsible for the highest mortality around the world with approximately one third of the world population is infected with Mycobacterium tuberculosis. It is reported that about 8 million new cases and 3 million deaths occur each year. Although the tuberculosis can be treated by using appropriate antibiotics, the majority failed in the treatment of tuberculosis is the late diagnosis of the disease in Egyptian patients and this is due to the lake of a simple and good effective diagnostic method. So that the control of the TB disease is based on early diagnosis and effective treatment of active cases.
There are several methods for diagnosis of tuberculosis. The popular detection method can be made by the microscopy or by the culture technique that are considered as a “gold standard”. The culture technique has advantage of distinguishing the Mycobacterium tuberculosis morphology from those of non- tuberculous Mycobacteria, but has a limitation of it require more time for results (6-8) weeks
There are newer techniques for diagnosis of tuberculosis such as polymerase chain reaction, restriction fragment length polymorphism (RFLP), DNA probes and BACTEC culture system. Those are sensitive and help in a rapid diagnosis. Some of which used recently but, most of them cannot be used in routine diagnosis as they are not cost effective and are not available everywhere.
Antibody production is a powerful diagnostic marker in many infectious cases. It is used in a serodiagnositic method like ELISA because of convenient sampling, low costs, easy to operate and rapid to determine.
According to the reports of the World Health Organization a serological test should possess sensitivity of over 80% and specificity of over 95% to replace the gold standard method. Until to date there is no tested antigen that has these features.
The current study aims to purify and test some immunologically important antigens from the culture filtrate proteins of M.TB and to evaluate the possibility of using these antigens as a good serodiagnostic tool for TB.
In the present study the culture filtrate proteins secreted by M.TB were isolated. The primary characterization of CFPs of M.TB was carried out by SDS-PAGE on the basis of molecular weight.
The secreted antigens 100KDa and 65KDa were purified from M.TB CFPS by preparative SDS-PAGE followed by passive chemical elution. Passive chemical elution has several advantages to be used as it doesn’t require complicated equipment, rapid elution with satisfactory proteins yield.
There was another antigen (recombinant RV2031) that was kindly provided by Prof.Dr/ Peter Andersen, Department of Infectious Diseases Immunology, Statens Serum Institute, Copenhagen, Denmark.
A checker board titration was performed for determination of the best serum dilution against 2µg/ml of each antigen and crude CFPs to be used in ELISA. The optimum serum dilution for 2 antigens (Rv2031 & 100 KDa) and crude CFPs was 1:400. However, the 65 KDa did not give regular results in checker board ELISA so, the same serum dilutions (1:400)was used.
The sensitivities of recombinant antigen Rv2031, 100 KDa. 65 KDa and crude CFPs were (90%, 85%, 20% & 75%) respectively with specificities (100%) for all tested antigens, and accuracies were (94%, 91%, 53% & 85%). In conclusion Rv2031 and 100 KDa may be considered as promising diagnostic markers of pulmonary tuberculosis but, more studies will be needed to evaluate the serodiagnostic potential, of the 65 KDa where it showed diminished accuracy (53%) confirmed by the fact that it has been reported to share common epitops with other organisms.
Finally, it is reported that there are two strategies that can be used to improve the specificity of TB serodiagnosis: firstly, always using the specific antigens present only in M. tuberculosis; secondly, avoiding possible antigen contamination during the purification process.
finally the present study where we used another method of diagnosis that is polymerase chain reaction to evaluate the role of IS6110 gene in diagnosis of tuberculosis where the DNA samples were simply extracted from blood of patients as well as healthy subjects without any purification step followed by samples amplification by PCR then detection by agrose gel electrophoresis.
This PCR technique showed sensitivity, specificity and accuracy of (64, 100 & 82%) respectively. In conclusion the tested PCR technique showed promising results based on an easy and simple DNA extraction method from blood samples which may be improved by using a DNA purification method.
At the end there are three approaches that can be taken into account to decrease the burden of TB in the future:
The first strategy is depending on the rapid diagnosis of persons with active TB and early introduction of effective treatment to prevent them from spreading Mycobacterium tuberculosis in the environment.
An alternative strategy is prophylactic vaccination against tuberculosis.
The third approach in the fight against tuberculosis is the detection and treatment of people with latent TB. This approach is aimed to prevent progression of latent TB to active TB.
Recommendations:
Based on these results we recommend that the recombinant (RV2031) and 100-KDa antigens may be used as a good diagnostic marker for tuberculosis in Egyptian patients.
It is not recommended to use 65 KDa antigen as a serodiagnostic marker for TB as it showed adiminished accuracy.
The tested PCR technique showed promising results based on an easy and simple DNA extraction method from blood samples which may be improved by using a DNA purification method.