الفهرس 
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Abstract
Infectious diseases remain the highest cause of death in the world. TB remains one of the world’s major causes of illness and death. In 1993, the World Health Organization (WHO) declared TB to be a global health emergency. One-third of the world’s population or two billion people carry the TB bacteria, more than 9 million of whom become sick each year with “active” TB which can be spread to others, and three million deaths are reported every year around the globe.
In most cases, M. tuberculosis infection is asymptomatic, latent infection that in some instances progress into TB infection. However, untreated active infections have more than 50% mortality rate.
The most powerful tools in any TB control program are prompt diagnosis and successful treatment of patients with active contagious disease. Early mycobacterial identification to the species level is important because it would help in the initiation of early and appropriate treatment of patients. However, identification of mycobacteria by conventional methods is time-consuming and not always conclusive.
Although the isolation of tubercle bacilli from clinical specimens is the gold standard for diagnosis, this may not be achievable in every single patient. Chest X-ray, identification of bacilli, and the histopathological detection of granulomatous lesions in addition to clinical findings generally lead to true diagnosis.
The sensitivity of routine smear-microscopy is approximately 50%, culture techniques take several weeks to yield results, and suitable representative biological samples are frequently unobtainable either due to lack of sputum production or poor sample quality. The HIV pandemic compounds this problem by increasing the incidence of smear-negative and sputum-scare TB.
Although tuberculin skin test (TST) has long been used for detection of both active and latent tuberculosis, it has a low specificity. The biggest drawback of TST is the cross-reaction with nontuberculous mycobacteria (NTM) or with Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) vaccine strains. Because protein-purified derivative (PPD) is a culture filtrate of tubercle bacilli containing over 200 antigens shared with the bacille Calmette-Gue´rin (BCG) vaccine and most nontuberculous mycobacteria, individuals vaccinated with BCG but not infected with MTB can test falsely positive using the tuberculin test.
Culture filtrate antigens play a role as targets of protective immune responses; this hypothesis has been supported by a number of studies in the mouse and guinea pig models of TB infection.
Many antigens are studied these days for their potential effect as candidate vaccine for tuberculosis or as immunodiagnostic reagent.
Identification and characterization of the some M. tuberculosis-specific antigens like ESAT-6 and CFP-10 has led to the development of new specific diagnostic tests for infection with M. tuberculosis. Novel interferon-(IFN)-γ release assays (IGRAs) provide distinct advantages. They are highly MTB-specific and thus not confounded in populations containing a high proportion of BCG-vaccinated individuals. They avoid boosting of immune responses by ex-vivo testing and possess logistical conveniences. Immune recognition of ESAT-6 is known to be highly specific for exposure to members of the TB complex, so it serves as a marker for prior M. tuberculosis infection.
To accomplish our aim in this study, we selected in this study 69 subjects divided into three clinical groups as follow. Group (1): Healthy subjects with negative TST results (n = 16); Group (2): Healthy subjects with positive TST results (n = 16), while Group (3): Patients with active pulmonary tuberculosis (n = 37).
We examined the immune responses of the healthy TST negative, healthy TST positive and patients with active pulmonary TB to M. tuberculosis culture filtrate protein, 38 kDa purified antigen and mixture of overlapping synthetic peptides mixture of early secretory antigen target 6 (ESAT-6). Our goal is to test the potential of ESAT-6 Pepmix as a candidate immunodiagnostic reagent to detect the infection with M. tuberculosis among Egyptian personals.
In this study, supernatants after overnight stimulation of whole blood with M. tuberculosis culture filtrate antigens, 38 kDa purified antigen and ESAT-6 overlapping synthetic peptides mixture were collected and immune response were determined by measuring the released interferon gamma (IFNγ) in the plasma by using ELISA as readout of T-cell activation and we were also measured interleukin-17 (IL-17) using ELISA technique to determine whether it could increase the sensitivity and specificity of this assay in diagnosing M. tuberculosis infection.
Our data showed that; a clear T cells response by measuring the released IFNγ in response to culture filtrate crude protein, 38 kDa purified antigen and ESAT-6 overlapping synthetic peptides mixture stimulation in the subjects under the study using IFNγ ELISA technique. With CF and 38 kDa stimulation it was found that there was no significant difference between the TB patients and the TST positive healthy controls in the overall T cells response and the cytokines measured. Unlike CF and 38 kDa, we found that ESAT-6 Pepmix stimulation was able to distinguish the TB patients and the TST positive healthy controls populations studied. Egyptian TB patients highly recognized ESAT6 Pepmix.
Following stimulation, active TB cases produced significantly lower levels of IL-17 compared with in IFNγ. Measurement of IL-17 yielded no statistically significant differences between the groups and IL-17 has no greater sensitivity than IFNγ in active TB patients.
So in conclusion; we can say that study confirmed that measurement of IFNγ levels after ESAT-6 stimulation raised the possibility of early diagnosis in the TB group. The IFNγ assay using ESAT-6 overlapping synthetic peptides mixture as an antigenic stimulus has the potential to be used as a tool for the immunodiagnosis of TB infection. Our results show that mixtures of synthetic overlapping peptides have potency equivalent to that of whole ESAT-6 for sensitive and specific detection of infection with M. tuberculosis, and peptides have the advantage of faster production at lower cost.