 Vol.2, No.3, 171-176 doi:10.4236/health.2010.23025 Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/ (2010) Health Rapid immunodiagnostic assays for Mycobacterium Tuberculosis infection Roba M. Talaat1*, Gamal S. Radwan2, Abdelaziz A. Mosaad3, Saleh A. Saleh4, Kalied Bassiouny1 1Department of Molecular Biology, Gen etic Eng i neering a nd Biotec hnology Research Institute (GEBRI), Minufiya University, Egypt; *Robamtalaat@yahoo.com 2Department of Molecular Diagnosis, Genetic Engineering and Biotechnolo gy Research Institute (GEBRI), Minufiya University, Egypt 3Department of Microbiology, Faculty of Veterinary Me dicine, Minufiy a University, Egypt 4Diagsera /VACSERA , Ministry of Health, Egypt Received 24 November 2009; revised 6 January 2010; accepted 8 January 2010. ABSTRACT Purpose: There is a need for a continued effort to develop rapid immunodiagnostic assays for tuberculosis (TB) infection with greater sensi- tivity and specificity that can be used in the field and in the laboratory and that can be formatted for use with multiple species. This would help to obtain definitive early diagnosis of TB. The present study was developed to determine the role of using early secreted antigenic target-6 (ESAT-6) in immunodiagnosis of Mycobacterium tuberculosis. Methods: Serum samples were obtained from TB infected patients and normal healthy controls. Two rapid immunodiagnostic assays (Enzyme-linked immunosorbent assay (ELISA) and Immunoblotting) were performed. Results: The sensitivity of immunoblotting assay was 100%; however, ESA T-6 antigen was not able to discriminate between patients and normal controls. Application of direct ELISA using ESAT -6 antigen yielded 97.6% sensitivity and 75% specificity for the diagnosis of TB infection. Conclusion: In conclusion, the detection of an- tibodies against ESAT-6 antigen in the sera of TB patients by direct ELISA could be used as a preliminary assay for diagnosis of human M. tuberculosis infection. A combination of the ELISA with either radiological or microscopic examination is required to overcome the low specificity of the assay for negative results. Keywords: Tuberculosis; Diagnosis; ESAT-6; ELISA; Immunoblotting 1. INTRODUCTION Tuberculosis (TB) is a global health problem with one- third of the world’s population latently infected with Mycobacterium tuberculosis (MTB) and about 8 million cases of active disease occurring each year [1,2]. Due to the increasing numbers of persons with MTB in the last years, the worldwide dissemination of HIV infection, the strongest risk factor for MTB development, and the con- tinual migration of people from areas with a high inci- dence of TB to highly industrialized countries, MTB is considered a global emergenc y [3-6]. In general, infection by MTB is controlled initially by host defenses, and the infection remains latent. However, latent TB infection has the potential to develop into active TB at any time. Be- cause active TB is infectious and leads to the spread of MTB, rapid diagnosis and effective treatment of indi- viduals with active TB are the m ost im portant com ponent of TB control programs. Moreover, identification and treatment of pe rsons with latent MTB infection who are at high risk of progressing to active disease, also may con- tribute to TB control [7]. Diagnosis of MTB based on clinical symptoms, chest radiography and sputum microscopy is sensitive but not specific [8]. Culture of bacteria is time-consuming, and usually the bacillus is not cultured [9], whereas nucleic acid-based methods such as polymerase chain reaction (PCR) are not consistently accurate enough for the di- agnosis of smear-negative pulmonary TB [10]. The tu- berculin ski n te st (TS T), usin g puri fie d pr ot ein deri vati ve (PPD), is largely used for both diagnosis and screening. The greatest drawback of PPD is its broad cross-reactivity with antigens derived from several mycobacterial species, e.g., attenuate d M. bovis bacillus Calmette-Gue´rin (BCG) used for vaccination, greatly decreasing the specificity of the TST [1 1,12]. Moreover , 10-25% of TB patients have a negative TST result, and this percentage increases up to 50% in patients with advanced disease or with immuno- deficiency due to HIV coinfection [13,14]. MTB infec- tion evokes a strong cel l -mediated immune response, and detection of specific T cells might be a mean to detect infection [15-17]. Because of their ease of performance and cost effectiveness, serodiagnostic tests constitute a
 R. M. Ta laat et al. / HEALTH 2 (2010) 171-176 Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/ 172 promise for early detection of TB. In fact, no serological test is commonly used in the diagnosis of TB [18]. In an effort to develop more accurate diagnostic tools, recent studies have led to the identification of the ge- nomic segment BCG-region 1 (RD1), present in MTB but absent from all strains as well as alm ost all environmental mycobacteria [19-21]. Therefore, RD1 gene products offer the potential for the development of new diagnostic tests that may differentiate MTB infection from BCG vaccination as well as exposure to environmental myco- bacteria. Two secretory, low molecular mass proteins, early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) have been identified as a product of this gene [22,23]. The antigens have already shown great potential for tuberculosis diagnosis [24,25]. Thus, the purpose of this study was to evaluate the di- agnostic potential of two rapid immunodiagnostic assays (Immunoblotting and Enzyme-linked immunosorbent assay (ELISA) for the detection of M. tuberculosis in- fections using ESAT-6 antigen. 2. MATERIALS AND METHODS 2.1. Human Sera Forty-two serum samples from patients with pulmonary tuberculosis were obtained from Mamora Chest Hospital (Alexandria, Egypt). They we re diagnose d by a specialist using smear and/or sputum culture (Ziehl-Neelsen stain) [26] and confirmed by chest X ray. Sixteen control sera were collected from laboratory staffs (who never suffer from TB infection and did not get a vaccination). All investigations were done in accordance with the Ministry of Health, health and human Ethical Clearance Com- mittee guidelines for clinical researches. Minufiya Uni- versity local ethics committee approved the study pro- to co l. All patients and controls agreed to be enrolled in this study. 2.2. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ESAT-6 antigen was kindly prepared and provided by Dr. William C. Davis (College of Veterinary Medicine, Washington State University, Pullman, WA, USA). Pro- tein content was estimated by the Brad ford method [27]. Antigen was analyzed by 12% SDS-PAGE using a dis- continuous SDS-PAGE system [28] and stained with Coomassie brilliant blue (Sigma Chemical Co., St. Louis, MO, USA). The protein molecular weight standard was from invitrogen (Invitrogen Corporation Carlsbad, CA, USA). 2.3. Immunoblotting ESAT-6 was subjected to 12% SDS-PAGE, electro- transferred onto 0.22µm nitrocellulose (NC) membrane (Bio-Rad Lab oratories, Ri chmond, CA, USA) [ 29]. Elec- trophoretic transfer was accom plished in 1h at 4°C with a constant 200V. After electrotransfer, the sheet was cut into 0.2cm wide strips. The resolved bands on NC mem- brane were visualized with specific immunological de- tection as described by [29-30]. Briefly, the antigen- containing strips were incubated with sera of MTB in- fected patients diluted 1:50 in blocking buffer (phos- phate-buffered saline (PBS) containing 5% non-fat milk and 0.3% pol yoxyethyle ne-20 (Twee n-20) (Sigm a) for 1h. All incubations were carried out at room temperature on an orbital shaker (Bellco, Vineland, NJ. USA). After incubation, the NC strips were washed with hot (65C) washing buffer (PBS/0.3%Tween-20) and incubated for 1h with peroxidase-labeled goat anti-human IgG (GAH- GPOD) (kindl y suppli ed by Dr. Victor Tsang, Divisio n of Parasitic Diseases, Centers for Disease Control and Pre- vention, Atlanta, GA) diluted 1:1000. The strips were then washed 3 times with washing buffer and once with PBS only. Reactive bands were visualized by incubating the NC strips with freshly prepared substrate solution [50mg of 3, 3'-diaminobenzidine (DAB) and 5µl H2O2 (30%) dissolved in 50ml PBS, pH 7.2) (Sigma Chemical Company, St. Louis, MO]. Positive reaction bands ap- peared within 10 min. The reaction was then stopped using distilled water. 2.4. Enzyme Linked Immunosorbent Assay (ELISA) The ELISA was performed in 96 well flat bottomed mi- croplates (Griener Labortechnik, Kremsmunste r, Austria). The optimum antigen, serum and conjugate concentra- tions and the incubation times were determined by check- erboard titration. The wells were coated with 100 l of ESAT-6 diluted in PBS (pH 7.2). The plates were incu- bated 1h at 37C then overnight at 4C. After one wash with PBS containing 0.05% Tween-20 (PBS/Tw), 200 l of 5% non-fat milk diluted in PBS/Tw were added to each well and incubated for 1.5 h at 37C. After one wash with PBS/Tw, tested TB-infected and normal healthy control sera (100 l/well) diluted in dilution buffer (PBS/Tw containing 1% non fat m ilk) were a dded in duplicates an d incubated at 37C for 1.5h. At the end of the incubation period, plat es were washed 3 tim es wit h PBS/Tw and 100 l GAHG-POD were incorporated to each well at dilution 1:1000 in dilution buffer. After 4 washes with PBS/Tw, 100 l of TMB (3, 3’, 5, 5’-tetramethyl benzidine) and H2O2 (0.02% in citric acid buffer) substrate (Kirkegrd and Perry Lab, Caithersuburg, MY, USA) were added to each well. The incubation time for the substrate was 15min. at room temperature. The optical density was measured at 620nm using the UV-max ELISA plate reader (Molecular Devices Corp.).
 R. M. Talaat et al. / HEALTH 2 (2010) 171-176 Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/ 173 2.5. Statistical Analysis Data are expressed as mean OD ± SD and were analyzed using the statistical software package for social science (SPSS). Suitable cutoff value for the ELISA was deter- mined by receiver operating characteristics (ROC) analysis. Comparisons of patients and normal control groups were performed by the Student’s t-test. The dif- ferences were considered significant if the probability was associated with p<0.05. 3. RESULTS 3.1. Gel Electrophoresis ESAT-6 was subjected to SDS-PAGE followed by Comassie brilliant blue staining. As the results in Figu r e 1 showed, 2 bands were identified in ESAT-6 (7.1 and 6.5 kDa). 3.2. Immunoblotting On screening of the TB infected sera, the im munoblotting assay sensitivity was 100% as all TB infected patients reacted with ESAT-6. However, the antigen was not able to discriminate between patients and normal controls (Figure 2). 4. ELISA Each serum sample was tested for its reactivities against ESAT-6. Figure 3 showed the reactivity of TB infected sera and uninfected controls against ESAT-6. The mean absorbance value in the TB patients was 0.566 0.017, significantly higher (p<0.01) than that in normal control Figure 1. Coomassie blue stained 12% SDS- PAGE gel of ESAT-6 antigen. Controls TB Pat ien ts Figure 2. Western blot profile of M. tuberculosis patients and normal controls. ESAT (0.1 µg/mm) were separated by 12% SDS-PAGE, electrotransferred onto NC sheets, cut into 0.2 cm identical strips and reacted with patients and normal human serum (last 8 strips) diluted 1:50 in PBS/0.3%Tween-20/5% non-fat milk. Arrow pointed to the reactive band (6 kDa). 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Opti cal Densi ty (OD650) Figure 3. Reactivity of sera from M. tuberculosis infected patients against ESAT-6 antigen (as shown by direct ELISA). ROC Curve 1 - Specificity 1.00.75.50.250.00 Sensitivity 1.00 .75 .50 .25 0.00 Figure 4. ROC curves for ELISA with ESAT-6. group (0.360 0.029). After ROC analysis (Figure 4), the area under the curve and standard error of the area under the c urve were 0. 961 (95% confi dence interval [C I], 0.918 to 1.005) and 0.022, respectively. Among different cutoff values from the ROC analysis, a cutoff value of 0.351 was selected, as the sensitivity of 97.6 % and specificity of 75% at the chosen cutoff was optimal for our ELISA. Out of 42 TB infected sera, only 1 serum sample showed a negative result at the cutoff value (1/42) (false negative). On the other hand, out of 16 normal
 R. M. Talaat et al. / HEALTH 2 (2010) 171-176 Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/ 174 controls, 4 serum samples showed a positive resu lt at the cutoff value (4/16) (false positive). 5. DISCUSSION Along with HIV and malaria, MTB has been declared a global enemy [31]. Delay in diagnosis is significant re- garding not only disease prognosis at the individual level but also transmission within the community and the productive rate of TB epidemic [32,33]. An effective TB control program requires early diagnosis and immediate initiation of treatment. To control tub erculosis (TB), it is still necessary to find diagnostic methods that are both more rapid to carry out and more sensitive than traditional methods (smear and culture) but which are simpler and less expensive than the new molecular diagnostic tests that are based on the amplification of nucleic acids. Se- rological methods, which seem to be the ideal choice, are usually based on the detection of free soluble circulating antibodies against mycobacterial antigens. Thu s, there is a need for a continued effort to develop rapid immuno- diagnostic assays with greater sensitivity and specificity that can be used in the field and in the laboratory and that can be formatted for use with multiple species. Several secreted antigens hav e been characterized, and their util- ity for diagnostic testing as well as their attributes as vaccine components have been exploited [34-36]. An antibody detection test can be developed into sev- eral formats depending on the membrane, antigen(s) coat- ing, and incubation technique. Common designs include ELISA and immunoblotting formats (commercial sero- logical). TST has many drawbacks, such as the need for patients to return for test reading, as well as variability and subjectivity in test application and reading. Most importantly, TST has low specificity as PPD, the antig en used for the test, is a mixture of mycobacterial antigens also present in non tuberculous mycobacteria and in the BCG vaccine strains [1 1]. Although both assays (TST and ELISA) do not offer a good specificity in detection of M. tuberculosis in fection, EL ISA has many other ad vantages over the TST. Objective quantitative results can be ob- tained the day after blood sampling, and time spent on return visits to have the TST read is spared. Furthermore, since no antigen is injected, the problem of a booster affect on sequential skin tests is avoided. The test is simple t o perform , and ca n be use d even i n count ries with less-developed infrastructure. ESAT-6 protein is secreted at an early or active phase of mycobacterial infection. It is virtually specific for M. tuberculosis complex representing a potential candidate for use in early detection as substitute or as improved skin test antigen [21-23,37-39]. Here in, we investigated the human antibody respon se against ESAT-6 antigen for the detection of M. tuberculosis infection using different immunoassays (immunoblotting and ELISA). Serial samples of sera collected from M. tuberculosis infected patients were subjected to our developed im munoblotting and ELISA to determine the serological response to ESAT6. Western blot analysis showed a reactive band at 6 kDa with all serum samples. This may suggest that, using immunoblotting format assay, ESAT-6 antigen is not only recognized with individuals with TB infection but also with normal healthy controls. This result merely reflects a case of cross reaction that may returned to shared epitope identification afforded by this assay . Thus, our work showed that western blotting using ESAT an- tigen is not the suitable choice for diagnosis of M. tu- berculosis infection and just used to support its diagnosis. On the ot her hand, applicati on of ELISA has i ncreased the sensitivity and specificity for diagnosis of M. tuber c ulosis infection to 97.6% and 75%, respectively. ESAT-6, for comparison, is broadly recognized early during disease in different species infected with M. tu- berculosis or M. bovis [37,40,41] and this antigen is generally reported to trigger the release of high levels of IFN- by sensitized peripheral blood mononuclear cells (PBMC) from TB patients [42,43]. This antigen dis- criminates TB patients from both BCG-vacci nated and M. avium patients and has therefore been suggested as a candidate for in vitro TB diagnosis [42,43]. The diag- nostic potential of low molecular weight culture filtrate antigens (ESAT-6 and CFP-10) of M. tuberculosis has been reported in earlier studies [24,25,44]. It has been previously repo rted th at subjects with latent TB infection may respond to ESAT-6 peptides and to the whole ESAT-6 protein [45-49]. Vincenti et al. [50] dem onstrated that, patients with active TB recognized the whole ESAT- 6 protein or PPD. The lack of response in some TB pa- tients may be due to their particular state of immune suppression [43-50]. Although serologic testing can detect specific anti- bodies to mycobacteria in serum and is attractive diag- nostic method due to its ease of application, they present low sensitivity and specificity due to the great heteroge- neity of the humoral response in patients with TB [51]. It is noteworthy that, in patients suffering from AIDS, in whom the number of T cells is decreased or even null, determining humoral response can be an invaluable tool in making an early diagno sis and gainin g epidemiological control over TB [52]. Thus, even with 75% specificity, getting 97.6% sensitivity with ESAT-6 in ELISA format has improved the diagnostic potential of this antigen for TB diagnosis. Imaz et al. [18] suggested the use of multi-antigen cocktail to improve the diagnostic utility of the ELISA. The design of antigen combinations would achieve the high diagnostic accuracy of the assay. In conclusion, this study offers the potential for de- veloping relatively rapid assays (ELISA and western blotting) for detection of M. tuberculosis infection. We have tested the application of antibody reactivity against ESAT-6 antigens as a serodiagnostic marker. The results showed that the use of ESAT-6 improves the diagnosis's
 R. M. Talaat et al. / HEALTH 2 (2010) 171-176 Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/ 175 sensitivity and provide an ELISA as a simple method that could be applied in TB diagnosis. To overcome the low specificity of the assay, a combination of the ELISA with either radiologi cal or mi croscopic examination is require- ed for negative results. 6. ACKNOWLEDGMENTS The current study was supported by Minufiya University and VACSERA. Authors would like to thank Dr. William C. Davis (Collage of Veterinary Medicine, Washington State University, Pullman, WA, USA) for his support by providing us with ESAT-6 antigen. REFERENCES [1] Brock, I., Weldingh, K., Leyten, E.M.S., Arend, S.M., Ravn, P. and Andersen, P. (2004) Specific T-Cell epitopes for immunoassay-based diagnosis of mycobacterium tu- berculosis Infection. Journal of Clinical Microbiology, 42(6), 2379-2387. [2] World Health Organization (WHO) (2007) Global tuber- culosis control: Surveillance, planning, financing. WHO report. Geneva: Wo rld Health Or ganization. WHO/ HTM/ TB/2007.376. [3] Di Perri, G., Cruciani, M., Danzi, M.C., Luzzati, R., De Checchi, G., Malena, M., Pizzighella, S., Mazzi, R., Solbiati, M., Concia, E., et al. (1989) Nosocomial epi- demic of active tuberculosis amo ng HIV infec ted pati ents. Lancet, 2, 1502-1504. [4] Lucas, S. and Nelson, A.M. (1994) Pathogenesis of tu- berculosis in human immunodeficiency virus-infected people. American Society for Microbiology Press, Wash- ington, D.C., 29. [5] Dye, C., Watt, C.J., Bleed, D.M., Hosseini, S.M. and Raviglione, M.C. (2005) Evolu tion of tuberculosis control and prospects for reducing tuberculosis incidence, pre- valence, and deaths globally. Journal of the American Medical Association , 293, 2767-2775. [6] Jasmer, R.M., Nahid, P. and Hopewell, P.C. (2002) Latent tuberculosis infection. New Engl and Journal of Medicine, 347, 1860-1866. [7] Bruchfeld, J., Aderaye, G., Palme, I.B., Bjorvatn, B., Britton, S., Feleke, Y., Kallenius, G. and Lindquist, L. (2002) Evaluation of outpatients with suspected pulmo- nary tuberculosis in a high HIV prevalence setting in Ethiopia: Clinical, diagnostic and epidemiological char- acteristics. Scandinavian Journal of Infectious Diseases, 34, 331-337. [8] Anderson, D., Anderson, V., Pentland, L., Sawyer, S., Starr, M. and Johnson, P.D. (2000) Attention function in secondary school stud ents receiving isoniazid prophylaxis for tuberculosis infection. Epidemiology and Infection, 124(1), 97- 101. [9] Sarmiento, O.L., Weigle, K.A., Alexander, J., Weber, D.J. and Miller, W.C. (2003) Assessment by meta-analysis of PCR for diagnosis of smear-negative pulmonary tuber- culosis. Journal of Clinical Microbiology, 41, 3233-3240. [10] Chaparas, S.D., Maloney, C.J. and Hedrick, S.R. (1970) Specificity of tuberculins and antigens from various spe- cies of mycobacteria. American Review of Respiratory Diseases, 101, 74-83. [11] Harboe, M. (1981) Antigens of PPD, old tuberculin, and autoclaved Mycobacterium bovi s BCG studied by crossed immunoelectrophoresis. American Review of Respiratory Diseases, 124, 80-87. [12] Huebner, R.E., Schein M.F. and Bass Jr.J. (1993) The tube r- culin sk in t est . Clinical Infectious Diseases, 17, 968-975. [13] Huebner, R.E., Schein, M.F., Hall, C.A. and Barnes, S.A. (1994) Delayed type hypersensitivity energy in human immunodeficiency virus-infected persons screened for infection with Mycobacterium tuberculosis. Clinical In- fectious Diseases, 19, 26-32. [14] Converse, P.J., Jones, S.L., Astemborski, J., Vlahov, D. and Graham, N.M. (1997) Comparison of a tuberculin interferon-gamma assay with the tuberculin skin test in high-risk adults: Ef fect of human immunodeficiency virus infection. Journal of Infectious Disease, 176, 144-150. [15] Stenger, S. and Modlin, R.L. (1999) T-cell mediated im- munity to Mycobacterium tuberculosis. Current Opinion in microbiology, 2, 89-93. [16] Flynn, J.L. and Chan, J. (2001) Immunology of tubercu- losis. Annual Review of Immunology, 19, 93-129. [17] Delgado, J.C., Tsai, E.Y., Thim, S., Baena, A., Boussiotis, V.A., Reynes, J.M., Sat h, S., Grosje an, P., Yunis, E.J. an d Goldfeld, A.E. (2002) Antigen-specific and persistent tuberculin energy in a cohort of pulmonary tuberculosis patients from rural Cambodia. Proceedings of the Na- tional Academy of Sciences, 99, 7576-7581. [18] Imaz, M.S., Schmelling, M.F., Kaempfer, S., Spallek, R. and Singh, M. (2008) Serodiagnosis of tuberculosis: Specific detection of free and complex-dissociated anti- bodies anti-mycobacterium tuberculosis recombinant an- tigens. Brazilian Journal of Infectious Diseases, 12(3), 234-244. [19] Mahairas, G.G., Sabo, P.J., Hickey, M.J., Singh, D.C. and Stover, C.K. (1996) Molecular analysis of genetic differ- ences between M ycobacterium bovis BCG and virulent M. bovis. Journal of Bacteriology, 178, 1274-1282. [20] Behr, M.A., Wilson, M.A., Gill, W.P., Salamon, H., Schoolnik, G.K., Rane, S. and Small, P.M. (1999) Com- parative genomics of BCG vaccines by whole genome DNA microarray. Science, 284, 1520-1523. [21] Arend, S.M., van Meijgaarden, K.E., de Boer, K., de Palou, E.C., van Soolingen, D., Ottenhoff, T.H. and van Dissel, J.T. (2002) Tuberculin skin testing and in vitro T cell responses to ESAT-6 and culture filtrate protein 10 after infection with mycobacterium marinum or M. kan- sasii. Journal of Infectious Disease, 186, 1797-1807. [22] Sorensen, A.L., Nagai, S., Houen, G., Andersen, P. and Andersen, A.B. (1995). Purification and characterization of a low-molecular-mass T-cell antigen secreted by My- cobacterium tuberculosis. Infection and Immunity, 63, 1710-1717. [23] Berthet, F.X., Rasmussen, P.B., Rosenkrands, I., Andersen, P. and Gicquel, B. (1998) A mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10). Microbiology, 144 (Pt. 11), 3195-3203. [24] Arend, S.M., Andersen, P., van Meijgaarden, K.E., Skjot, R.L., Subronto, Y.W., van Dissel, J.T. and Ottenhoff, T.H. (2000) Detection of active tub erculosis infection by T cell responses to early-secreted antigenic target 6-kDa protein and culture filtrate protein 10. Journal of Infectious Dis- ease, May, 181(5), 1850-1854. [25] Ewer, K., Deeks, J., Alvarez, L., Bryant, G., Waller, S., Andersen, P., Monk, P. and Lalvani, A. (2003) Compari-
 R. M. Talaat et al. / HEALTH 2 (2010) 171-176 Copyright © 2010 SciRes http://www.scirp.org/journal/HEALT / Openly accessible at H 176 son of T-cell-based assay with tuberculin skin test for di- agnosis of Mycobacterium tuberculosis infection in a school tuberculosis outbreak. Lancet, 361, 1168-1173. [26] Crookham, J. and Dapson, R. (1991) Hazardous chemi- cals in the histopathology laboratory, 2nd ED, Anatech. [27] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein util- izing the principle of protein dye binding. Analytical Biochemistry, 72, 248-254. [28] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bactriophage T4. Na- ture, 277, 680-685. [29] Towbin, M., Staehelin, T. and Gordon, J. (1979) Electro- phoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and applications. Pr ocee- dings of the National Academ y of Sciences, 76, 4 350-435 4. [30] Burnette, W.N. (1981) “Western Blotting”: Electropho- retic transfer of proteins from SDS-PAGE to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein. Analytical Biochemistry, 11 2, 195-203. [31] Storla, D.G., Yimer, S. and Bjune, G.A. (2008) A system- atic review of delay in the diagnosis and treatment of tu- berculosis. BMC Public Health, 8, 15. [32] World Health Organization (WHO) (2005) Global tuber- culosis control: Surveillance, planning, financing. WHO report. Geneva: World Health Organization. WHO/HTM/ TB/2005.349. [33] Bjune, G. (2005) Tuberculosis in the 21st century: An emerging pandemic? Norsk Epidemiologi, 15(2), 133-139. [34] Bothamley, G.H. (1995) Serological diagnosis of tuber- culosis. European Respiratory Journal (Supplement 20), 676s-688s. [35] Lodha, R. and Kabra, S.K. (2004) Newer diagnostic mo- dalities for tuberculosis. Indian Journal of Pediatrics, 71(3), 221- 227.Links. [36] Orme, I.M. (2006) Preclinical testing of new vaccines for tuberculosis: A comprehensive review. Vaccine, 9;24(1), 2-19. [37] Pollock, J.M. and Andersen, P. (1997) The potential of the ESAT-6 antigen secreted by virulent mycobacteria for specific diagnosis of tuberculosis. Journal of Infectious Disease, 175, 1251-1254. [38] Harboe, M., Oettinger T., Wiker H.G., Rosenkrands I. and Andersen P. (1996) Evidence for occurrence of the ESAT-6 protein in Mycob acterium tuberc ulosis and virulent Mycobacterium bovi s and for i ts absence in Mycobacte rium bovis BCG. Infection and Immunity, 64, 16-22. [39] Geluk, A., van Meijgaarden, K.E., Franken, K.L., Sub- ronto, Y.W., Wieles, B., Arend, S.M., Sampaio, E.P., de Boer , T., Faber , W.R., Naafs, B. and Ottenhoff, T.H. (2002) Identification and characterization of the ESAT-6 homo- logue of Mycobacterium leprae and T-cell cross-reactivity with Mycobacter ium tuberculosis . Infection and Immunity, 70, 2544-2548. [40] Andersen, P., Andersen, A.B., Sorensen, A.L. and Nagai, S. (1995) Recall of long-lived immunity to Mycobacte- rium tuberculosis infection in mice. Journal of Immu- nology, 154, 3359-3372. [41] Brandt, L., Oettinger , T., Holm, A. and Andersen, P. (1996) Key epitopes on the ESAT-6 antigen recognized in mice during the recall of protective immunity to Mycobacte- rium tuberculosis. Journal of Immunology, 157, 3527- 3533. [42] Ulrichs, T., Munk, M. E., Mollenkopf, H., Behr-Perst, S., Colangeli, R., Gennaro, M. L. and Kaufmann, S.H. (1998) Differential T cell responses to Mycobacterium tubercu- losis ESAT-6 in tuberculosis patients and healthy donors. European Journal of Immunology, 28, 3949-3958. [43] Ravn, P. , Demissie, A., Egua le, T . , W o ndwosson, H., Lein, D., Amoudy , H.A., Mustafa, A.S., Jensen, A.K., Holm, A., Rosenkrands, I., Oftung, F., Olobo, J., von Reyn, F. and Andersen, P. (1999) Human T cell responses to the ESAT -6 antigen from Mycobacterium tuberculosis. Journal of Infectious Disease, 179(3), 637-645. [44] Laurens, A., Van Pinxteren, H., Ravn, P., Agger, E.M., Pollock, J. and Andersen, P. (2000) Diagnosis of tuber- culosis based on the two specific antigens ESAT-6 and CFP10. Clinical and Diagnostic Laboratory Immunology, 7(2), 155-160. [45] Lalvani, A., Pathan, A.A., Durkan, H., Wilkinson, K.A., Whelan, A ., Deeks, J .J., Reece, W. H., Latif, M ., Pasvol, G. and Hill, A.V. (2001a) Enhanced contact tracing and spa- tial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Lancet, 357, 2017-2021. [46] Lalvani, A., Nagvenkar, P., Udwadia, Z., Pathan, A.A., Wilkinson, K.A., Shastri , J .S., Ew er, K., Hill, A.V., Mehta, A. and Rodrigues, C. (2001b) Enumeration of T cells specific for RD1-encoded antigens suggests a high pre- valence of latent Mycobacterium tuberculosis infe- ction in healthy urban Indians. Journal of Bacteriology, 183, 469-477. [47] Lalvani, A., Pathan, A.A., McShane, H., Wilkinson, R.J., Latif, M., Conlon, C.P., Pasvol, G. and Hill, A.V. (2001c) Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. American Journal of Respiratory and Critical Care Medicine, 163, 824-828. [48] Pathan, A.A., Wilkinson, K.A., Klenerman, P., McShane, H., Davidson, R.N., Pasvol, G., Hill, A.V. and Lalvani, A. (2001) Direct ex vivo analysis of antigen-specific IFN-γ-secreting CD4 T cells in Mycobacterium tubercu- losis-infected individuals: Association with clinical dis- ease state and ef fe ct of treatm ent. Journal of Immunology, 167, 5217-5225. [49] Chapman, A.L., Munkanta, M., Wilkinson, K.A., Pathan, A.A., Ewer, K., Ayles, H., Reece, W.H., Mwinga, A., Godfrey-Faussett, P. and Lalvani, A. (2002) Rapid detec- tion of active and latent tuberculosis infection in HIV-positive individuals by enumeration of Mycobacte- rium tuberculosis-specific T cells . AIDS, 16, 2285-2293. [50] Vincenti, D., Carrara, S., De Mori, P., Pucillo, L.P., Pet- rosillo, N., Palmieri, F., Armignacco, O., Ippolito, G., Girardi, E., Amicosante, M. and Goletti1, D. (2003) Identification of early secretory antigen target-6 epitopes for the immunodiagnosis of active tuberculosis. Molecu- lar Mmedicine, 9(3), 105-111. [51] L yashchenko, K., Colang eli, R., Houde, M., Al Jahdali, H., Menzies, D. and Gennaro, M.L. (1998) Heterogeneous antibody responses in tuberculosis. Infection and Immu- nology, 66(8), 3936-3940. [52] Teixeira, H.C., Abramo, C. and Munk, M.E. (2007) Im- munological diagnosis of tuberculosis: Problems and strategies for success. Jornal Brasileiro de Pneumologia, 33(3), 323-334.
|