الفهرس 
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Abstract
Tuberculosis (TB), one of the oldest recorded human afflictions, is still one of the biggest killers among the infectious diseases, despite the worldwide use of alive attenuated vaccine and several antibiotics. It is believed that one third of the global population is infected with bacteria of the Mycobacterium tuberculosis (M.TB) complex, the causative agent of tuberculosis. More than 8 million new cases of tuberculosis occur annually leading to 2 million deaths.
The recommendation for the control of tuberculosis emphasize case detection so as to allow treatment of patients and there by limit transmission of the bacilli.
The simplest rapid method for TB diagnosis is the detection of acid-fast bacilli in sputum by microscopy. However, 40 to 60% of patients with pulmonary disease are sputum negative, and in this situation even contemporary culture methods take several weeks to become positive. Therefore, a number of alternative diagnostic tests that use molecular, immunological methods have been developed. While molecular methods overcome the insensitivity of the smear method and time required for culture, they depend upon retrieval of a specimen from the site of infection. This is often difficult in cases of tuberculosis in children. Rapid serological diagnostic tests such as the Enzyme-Linked Immunosorbent Assay (ELISA) and membrane chromatography tests, in contrast, are simple and inexpensive. A major problem encountered in serological techniques is the specificity and reactivity of antigens used.
According to the recommendations of the World Health Organization serological test should possess sensitivities of over 80% and test specificities of over 95%, to replace the ”gold standard” culture technique.
The present study aims to rapid diagnosis of tuberculosis in Egyptians patients using PCR dot blot and compare it with performance of PCR agarose gel.
To accomplish our aim in this study, 50 subjects were divided into four groups as follow: Group I consists of 10 healthy individuals; Group II consists of 20 patients with active pulmonary tuberculosis and show smear positive; Group III consists of 19 patients show smear negative; Group IV consists of 1 patient with extrapulmonary tuberculosis.
Samples were treated with DTT which liquefied the samples and the DNA was extracted by using the qiagen kit.
PCR was performed for all groups using IS6110 primers and analysis PCR product using agarose gel with ethedium bromide staining. There were 20 samples from 39 positive result on agarose gel for pulmonary TB patients (12 Ziehl Neelsen smear positive and 8 smear negative) and one sample for extrapulmonary TB patient which PCR-AG shows sensetivity and specifity 52.5% and 100% respictivity. The results obtained in this study demonstrate that in-house PCR is not an ideal tool for the diagnosis of tuberculosis under the present conditions in developing countries: sensitivity is unsatisfactory. The number of organisms in saliva and sputum samples analyzed might be a significantly lower value than would be found in ‘real’ samples, and therefore the ability to detect these organisms could be underestimated.
Data showed that causes of negative PCR results in culture-positive specimens were low number of bacteria, uneven distribution of Mycobacterial DNA in test sample, and the fact that M. tuberculosis bacilli have a tendency to form clumps and cords. It is also important to remember that the volume of samples used for PCR is much less than that used for culture. This may affect the sensitivity of PCR especially in samples with low bacterial counts.
Our goal is to know the sensitivity and specifity of PCR dot blot for the clinical groups in this study and compare it with PCR agarose gel.
Our results showed that the sensitivity of PCR agarose gel showed lower sensitivity as compared to PCR dot blot hyberdization.
PCR dot blot hyberdization showed positive results for all groups in this study except healthy groups which showed sensitivity 100%. Also showed that PCR dot-blot associated with a high clinical suspicion may provide an important contribution to the diagnosis of smear negative pulmonary tuberculosis mainly in patients that have not been previously treated.
For the detection of PCR products, dot blot hybridization confers several advantages over conventional ethidium bromide- stained gel analysis: it is less cumbersome, cost-effective, quicker, has fewer safety hazards, promotes objectivity in the reading of results and is suitable for use in epidemiological studies for the analysis of a large number of samples; the assay has been shown to be comparable in sensitivity and specificity to the other methods. Because of its high degree of specificity, PCR dot blot hybridization assay could be a confirmatory tool for establishing the presence of MTB in AFB-positive specimens and in AFB-negative specimens. However, to become a general purpose screening test, it would require improved the reliable detection of specimens containing low numbers of MTB.