Diagnosis of Mycobacterium Tuberculosis


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Diagnosis of Mycobacterium Tuberculosis

  1. 1. Biochemical Studies on Mycobacterium Tuberculosis Antigen Thesis Submitted for the degree of PhD in Biochemistry By Mohamed Mostafa Omran Biotechnology Research Center, New Damietta, Egypt Chemistry Department Faculty of Science Cairo University 2006
  2. 2. Approval sheet for submission Title of (Ph. D) thesis: Biochemical Studies on Mycobacterium Tuberculosis Antigen Name of candidate : Mohamed Mostafa Omran This thesis has been approved for submission by the supervisors Prof. Dr. Sanaa Osman Abdallah …………………………….. Professor of Organic Chemistry, Faculty of Science, Cairo University Prof. Dr. Abdelfattah Mohamed Attallah………………………… Professor of Immunology and Genetics, Director of Biotechnology Research Center, New Damietta Dr. Amr Saad Mohamed ……………………………………….. Assistant Professor of Biochemistry Faculty of Science, Cairo University Prof. Dr. Rifaat Hassan Hilal Chairman of Chemistry Department Faculty of Science- Cairo University
  3. 3. ABSTRACT Name: Mohamed Mostafa Omran Title of thesis: Biochemical Studies on Mycobacterium Tuberculosis Antigen Degree (Ph. D) thesis, Faculty of Science, Cairo University, 2006 The identification of tuberculosis (TB) antigen is a critical step toward accurate diagnosis of TB. Here, a target TB antigen was identified in serum, ascetic fluid and CSF samples from individuals with extra-pulmonary tuberculosis using specific monoclonal antibody and Western blot. The TB antigen was purified and characterized as protein of 55-kDa. The dot-ELISA detected the TB antigen in 90% sera of patients with extra-pulmonary TB and in 87% sera of patients with pulmonary TB with high degree of specificity (97%) among control individuals. In conclusion, the TB antigen detection immunoassay can be routinely employed to support clinical diagnosis of TB infection. Key words: Tuberculosis, Diagnosis, antigen, 55-kDa, Serum Supervisors: Prof. Dr. Sanaa Osman Abdallah, ……………………………... Prof. Dr. Abdelfattah Mohamed Attallah, ……………………………... Dr. Amr Saad Mohamed ……………………………... Prof. Dr. Rifaat Hassan Hilal Chairman of Chemistry Department Faculty of Science- Cairo University
  4. 4. ‫ﺑﺴﻢ اﷲ اﻟﺮﲪﻦ‬ ‫اﻟﺮﺣﯿﻢ‬ ‫"رب اﺷﺮح ﱃ ﺻﺪرى‬ ‫وﯾﺴﺮﱃ أﻣﺮى‬ ‫واﺣﻠﻞ ﻋﻘﺪة ﻣﻦ‬ ‫ﻟﺴﺎﱏ ﯾﻔﻘﻬﻮا‬ ‫ﻗﻮﱃ"‬ ‫ﺻﺪق اﷲ اﻟﻌﻈﯿﻢ‬
  5. 5. Acknowledgments I wish to express my gratitude to Prof. Dr. Sanaa Osman Abdallah, Professor of Organic Chemistry, Faculty of Science, Cairo University for her kind supervision, invaluable revision, valuable time and continuous advices which helped me to overcome many difficulties during the study. Gratefully, I would like to owes great thanks to Prof. Dr. Adbelfattah Mohamed Attallah, Professor of Immunology and Genetics, Director of Biotechnology Research Center (BRC), New Damietta, who deserves more thanks than I can give. He kindly suggested the point of this research and offered me all facilities with great help in designing the experiments, close supervision, revision and valuable advices during the study. My deepest thanks and gratitude are due to Dr. Amr Saad Mohamed, Assistant Professor of Biochemistry, Faculty of Science, Cairo University for his kind supervision, invaluable revision and valuable advice during the study. My deepest thanks and gratitude are due to Dr. Ahmed Abo Nagla, Assistant Professor of Chest, Faculty of Medicine, Al Azhar University, Cairo for kind help and providing the samples for the study. Finally, all this work has been financially supported completely and carried out at BRC, New Damietta, Egypt and I would like to thank Dr. Hisham Ismail, Senior of Immunochemistry, Research & Development Department, for his invaluable assistances, comments, and continuous encouragement during the study and I would like to thank all my colleagues at BRC especially Dr. Gellan Ibrahim, for her kind help and I would like to thank everyone who gives me a hand throughout this study. Mohamed M. Omran 2006
  6. 6. To my Loved Parents, To my Dear brothers Dr. Tarek, Aded El Hamid and Ahmed, To my Dear Sister Azza, To my Beloved and Supportive Wife Entessar, To my Beloved Son Mostafa. To my Beloved daughter Azza.
  7. 7. Note Several parts of this thesis of the candidate Omran M has been published in the following international journals: 1. Attallah AM, Abdel Malak CA, Ismail H, El-Saggan AH, Omran MM, Tabll AA. 2003. Rapid and simple detection of a Mycobacterium tuberculosis circulating antigen in serum using dot-ELISA for field diagnosis of pulmonary tuberculosis. J Immunoassay Immunochem, 24: 73-87. 2. Attallah AM, Osman S, Saad A, Omran M, Ismail H, Ibrahim G, AboNaglla A. 2005. Application of a circulating antigen detection immunoassay for laboratory diagnosis of extra-pulmonary and pulmonary tuberculosis. Clin Chim Acta, 356: 58-66.
  8. 8. List of abbreviations ADA Adenosine deaminase activity AFB Acid fast bacilli AIDS Acquired immune deficiency syndrome BCG Bacillus Calmette - Guerin BCIP 5-Bromo-9-Chloro-3-Indolyl Phosphate BSA Bovine serum albumin CE Capillary electrophoresis CMI Cell-mediated immunity CSF Cerebrospinal fluid CT Computed tomography DTH Delayed-type hypersensitivity ECM Extracellular matrix ELISA Enzyme linked immunosorbent assay HAT Hypoxanthin aminopterin thymidine HPLC High performance liquid chromatography HPRT Hypoxanthin guanine phosphoribosyl transferase INF Interferon kDa Kilo dalton KV Kilo volt LJ Lowenstein - Jensen medium M Mycobacterium mAb Monoclonal antibody NAA Nucleic acid amplification NBT Nitro blue tetra-zolium NC Nitrocellulose PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline i
  9. 9. PCR Polymerase chain reaction PNB Para - nitrobenzoic acid PPD Purified protein derivative PPD-S Purified protein derivative florescence Seibert RIA Radioimmunoassay RT Room temperature SDS- Sodium dodecyl sulfate- polyacrylamide gel PAGE electrophoresis TB Tuberculosis TBS Tris buffered saline TCA Tricholoroacetic acid TEMED Tetra ethylene diamine Tu Tuberculin units UV Ultraviolet V Voltage WHO World Health Organization ZN Ziehl – Neelsen stain ii
  10. 10. Contents Title Page I. Introduction and Aim of work 1 II. Review of literature 3 1. Mycobacterium tuberculosis 3 1.1. Morphology 4 1.2. Staining properties 4 1.3. Sensitivity to physical and chemical agents 5 1.4. Animal pathogenicty 6 1.5. Constituents of tubercle bacilli 6 7 2. Tuberculosis 2.1. Epidemiology of tuberculosis 7 2.2. Risk factors 8 2.3. Pathology of tuberculosis 10 2.3.1. Pulmonary tuberculosis 10 2.3.2. Extra-pulmonary tuberculosis 12 Types of extra pulmonary tuberculosis 13 Lymph node tuberculosis 13 Tuberculosis peritonitis 13 Genitourinary tuberculosis 14 Orthopedic tuberculosis 15 Miliary tuberculosis 16 Tuberculosis of the central nervous system 17 Tuberculosis of sinusitis 19 Other sites 19 2.4. Control of tuberculosis 19 21 3. Diagnosis of tuberculosis 3.1. Microscopic method 21 iii
  11. 11. Title Page 3.1.1. Ziehl - Neelsen staining technique 21 3.1.2. Auramine phenol fluorochrome staining technique 24 Biopsies 25 3.2. X – ray 25 3.3. Culture 26 3.4. Tuberculin skin testing 27 3.5. Adenosine deaminase activity 29 3.6. Serodiagnosis of tuberculosis 30 3.7. Polymerase chain reaction 33 3.7. 1. Nucleic acid amplification 33 3.7. 2. Ligase chain reaction 34 3.8. Mycobacterial antigen detection 3.8.1. Application of monoclonal antibodies in diagnosis of M. tuberculosis antigen 34 35 III-Material and methods 38 1. Samples 38 1.1. Serum samples 38 1.2. Cerebrospinal fluid 38 1.3. Tuberculous ascetic fluid 39 1.4. Bacilli Calmette-Guerin 39 2. Monoclonal antibody 39 3. Protein content determination 40 4. Sodium dodocyl sulphate-polyacrylamide gel electrophoresis 43 5. Immunoblotting technique 46 6. Purification of the 55 kDa antigen 48 7. Capillary electrophoresis 50 8. Biochemical characterization of the 55 kDa antigen 52 iv
  12. 12. Title Page 9. Amino acid analysis 55 10. Dot-ELISA 56 11. Statistical Analysis 57 IV. Results 59 V. Discussion 109 VI. Summary 131 VII. References 136 VII. Arabic summary v
  13. 13. List of figures Title Fig. no. 1 2 Epidemiology of TB in the world Mycobacterium tuberculosis stained with ZiehlNeelsen staining page 9 23 3 Standard calibration curve of bovine serum albumin 42 4 SDS-PAGE and western blot analysis of BCG 60 5 Relation between Rf values of unknown antigens and of standards protein mixture 63 Coomassie blue stained SDS-PAGE of sera from 6 pulmonary tuberculosis patients and non infected 65 individuals under reducing conditions Immunoblots of TB-55 mAb target antigen in sera of 7 pulmonary tuberculosis patients and non infected 66 individuals Coomassie blue stained SDS-PAGE of sera from 8 extra-pulmonary tuberculosis patients and non 68 infected individuals under reducing conditions Immunoblots of TB-55 mAb target antigen in sera of 9 extra pulmonary tuberculosis patients and non 69 infected individuals Coomassie blue stained SDS-PAGE of CSF from 10 tuberculous meningitis patients and non-tuberculous 71 CSF under reducing conditions Immunoblots of TB-55 mAb target antigen CSF from 11 tuberculous meningitis patients and non-tuberculous CSF vi 72
  14. 14. Title Fig. no. page Coomassie blue stained SDS-PAGE of tuberculous 12 ascetic fluid from peritonitis tuberculosis patients and non-tuberculous ascites fluid under reducing 74 conditions Immunoblots of TB-55 mAb target antigen in 13 tuberculous ascetic fluid from peritonitis tuberculosis 75 patients and non-tuberculous ascites fluids 14 15 16 17 18 19 20 Coomassie stain SDS-PAGE of purified 55 kDa antigen from pulmonary tuberculosis sera Coomassie stain SDS-PAGE of purified 55 kDa antigen from extra-pulmonary tuberculosis sera Coomassie stained SDS-PAGE of purified 55 kDa antigen from CSF Coomassie stain SDS-PAGE of purified 55 kDa antigen from ascites. Capillary electrophoresis (CE) electropherogram of purified 55 kDa antigen Reactivity of the purified 55 kDa antigen against TB55 monclonal antibody using dot-ELISA The relative percentages of the amino acid concentrations of the purified 55 kDa antigen vii 77 78 79 80 81-84 86 91
  15. 15. Title Fig. no. 21 22 Types of tuberculosis page 93 Dot-ELISA of serum samples from tuberculosis patients and non-infected individuals 96 Levels of circulating 55-kDa antigen detection using 23 Dot- ELISA in serum samples of pulmonary 98 tuberculosis patients Levels of circulating 55-kDa antigen detection using 24 Dot- ELISA in serum samples of extra-pulmonary 103 tuberculosis patients Overall levels of circulating 55-kDa antigen detection 25 using Dot- ELISA in serum samples of pulmonary 106 and extra-pulmonary tuberculosis patients 26 Advantages of circulating 55-kDa antigen detection by using Dot- ELISA in 506 serum samples viii 108
  16. 16. List of tables Table no. 1 2 Title Some available antibody tests for diagnosis of pulmonary Tuberculosis Rf values of unknown antigens and of standards protein page 32 62 mixture 3 4 5 Partial biochemical nature of the purified 55-kDa antigens reactive epitope Amino acid concentrations of the purified 55 kDa antigen The types of extra-pulmonary tuberculosis according to sites of infection in 93 serum samples 88 90 94 Advantages of circulating 55-kDa antigen detection by 6 using Dot-ELISA in serum samples of pulmonary 98 tuberculosis 7 Detailed analysis of extra-pulmonary tuberculosis using dot ELISA 102 Advantages of circulating 55-kDa antigen detection by 8 using Dot-ELISA in serum samples of extra-pulmonary 104 tuberculosis 9 Advantages of circulating 55-kDa antigen detection by using Dot- ELISA in serum samples ix 107
  17. 17. Introduction and Aim of work Introduction and Aim of work Tuberculosis (TB) is one the greatest causes of mortality worldwide (Costa et al., 2005; Kehinde et al., 2005 and Nahid et al., 2006). The World Health Organization (WHO) estimates that there are more than 8 million new cases of tuberculosis each year, 3 million deaths from the disease each year, and that one-third of the world population is infected with Mycobacterium tuberculosis and at risk for active disease (Philip, 2003). Although the lung is the primary site of tuberculosis in 80 to 84 %, extra-pulmonary tuberculosis has become more common with the advent of HIV infection (Martin et al., 2001; Liberato et al., 2004). The most commonly reported extra-pulmonary sites of disease are the lymph nodes (Kidane et al., 2002; Marais et al., 2006) pleura (Hiraki et al., 2004) and bones or joints (Dursun et al., 2003; Marmor et al., 2004 and N'dri et al., 2004). Other sites include the genitourinary system (Chavhan et al., 2004; Kulchavenya and Khomyakov, 2006), the central nervous system (Sutlas et al., 2003 and Thwaites et al., 2004), the abdomen (Balian et al., 2000; Sabetay et al., 2000 and Vardareli et al., 2004) and in rare cases, virtually any other organ (Gülgün et al., 2000; Chen et al., 2004 and Sethi et al., 2006). Efforts to control tuberculosis are currently hampered by the lack of effective tools for the detection of infected individuals, although new diagnostic tests are being developed (Walsh and McNerney, 2004; Nahid et al., 2006). In active pulmonary tuberculosis, clinical symptoms confirmed by a laboratory test give a relatively clear result, whereas diagnosis can be rather problematic in patients with extra-pulmonary tuberculosis (Blanie et al., 2005). Bacteria in extra-pulmonary tuberculosis cases can be present in low numbers at inaccessible sites (Martin et al., 2001). Despite rapid advances in molecular genetics for detection of M. tuberculosis, it is clear that interest in serodiagnosis 1
  18. 18. Introduction and Aim of work remains high, especially for those situations in which a specimen may not contain the infecting agent in particular in extra-pulmonary tuberculosis (Brodie and Schluger, 2005). Extensive efforts to devise a sensitive and specific serodiagnostic test for the detection of M. tuberculosis-circulating antigen have been made by several authors (Khomenko et al., 1996 and Stavri et al., 2003). Monoclonal antibodies provide a useful tool for the specific identification of Mycobacterium (Kolk et al., 1984 and Daniel et al., 1988). Recently, Attallah et al (2003) developed a simple and rapid dot-ELISA based on the detection of a 55-kDa TB antigen for field diagnosis of pulmonary tuberculosis using TB 55 monoclonal antibody (mAb). Aim of work: The present study aimed to identify the 55-kDa circulating M. tuberculosis antigen in different body fluids and evaluated the application of the developed dot-ELISA for the detection of target Mycobacterial antigen in serum samples of patients with pulmonary and extra-pulmonary tuberculosis. 2
  19. 19. Review of literature 1. Mycobacterium tuberculosis Mycobacteria are rod shaped bacteria that do not form spores. The genus Mycobacterium was first described in 1896 by Lehmann and Neumann and includes M. leprae, the leprosy bacilli, and M. tuberculosis (Grange, 2002). Robert Koch first isolated the causative agent of M. tuberculosis in 1882 by inoculating material from human cases of TB onto solidified serum and noting the development of tiny colonies of bacteria (Koch, 1882 and Gradmann, 2006). These had the characteristic staining properties of organisms he had already demonstrated in TB tissue. Injection of these bacteria into guinea-pigs caused classical TB and Koch was able to re-isolate in pure culture the same organisms from the guinea-pig tissue, thus fulfilling his postulates. In the following years many more species of Mycobacterium were described, including M. bovis, the bovine tubercle bacillus (Grange, 2002). At first Koch did not accept that M. bovis could infect humans, but eventually DNA studies showed that they have a greater than 95% homology and can therefore be considered to be variants of the same species. Mycobacterium are rod shaped aerobic bacteria that do not form spores. The name of the genus, Mycobacterium (fungus bacterium) is an allusion to the mould like pellicles formed when members of the genus are grown in liquid media (Grange, 2002). This hydrophobic property is due to their possession of thick and waxy cell wall. Although they do not stain readily once stained they resist decolorization by acid or alcohol after staining with hot carbol fuchsin or other aryl methane dyes and are therefore called acid fast bacilli. TB is a chronic granulomatous disease affecting human and many other mammals. It is caused by four closely related species M.tuberculosis (the human tubercle bacillus), M. bovis (the bovine tubercle bacillus), M. microti (the vole tubercle bacillus) and M. africamum (Grange, 2002). Although M .tuberculosis is the most common infection in humans, M. bovis is responsible for an 3
  20. 20. Review of literature increasing proportion of human TB cases (Cosivi et al., 1998; Bengis, 2000 and Kathleen et al., 2002) but some cases are due to M. bovis that is the principle cause of TB in cattle and many other mammals. The name M. Africamum is given to tubercle bacilli with rather variable properties and which appears to be intermediate form between the human and bovine types. It causes human TB and is mainly found in Equatorial Africa. M. microti which is very rarely if ever encountered nowadays is a pathogen of voles and other small mammals but not of human (Grange, 2002). 1.1. Morphology Members of the M. tuberculosis complex (tubercle bacilli) are non-motile non-sporing, non-capsulate, straight or slightly curved rods about 3 x 0.3 µm in size. In sputum and other clinical specimens they may occur singly or in small clumps, and in liquid culture they often grown as twisted rope-like colonies termed serpentine cords (Grange, 2002). 1.2. Staining properties Tubercle bacilli are difficult to stain with the Gram stain although they are usually considered to be Gram positive; staining is poor and irregular because of failure of the dye to penetrate the cell wall (Grange, 2002). The acid fastness of the Mycobacterium is attributable to their lipid content and to the physical integrity of the cell wall. The best explanation of the acid fastness of Mycobacterium is based on the lipid-barrier principle according to which an increased hydrophobicity of the surface layers follows the complexing of dye with mycolic acid residues that are present in the cell wall. This prevents exit of carbol fuchsin that has become trapped in the interior of the cell. Once stained by an aniline dye such as carbol fuchsin they resist decolorization with acid and alcohol and are thus termed acid and alcohol fast bacilli. This is generally shortened to acid fast bacilli or AFB (Selvakumar et al., 2005). In virtually all 4
  21. 21. Review of literature other bacteria the dye is removed by the acid-alcohol wash and the cells take up the counter stain–usually methylene blue or malachite green. The AFB are then seen as red bacilli on a blue green background composed of an interlacing layer of lipids, peptidoglycans and arabinomannans. The aniline dye forms a complex with this layer and is held fast despite the action of the acid-alcohol. This allows the detection of AFB in specimens using a simple staining technique described by Ziehl in 1882 and modified by Neelsen in 1883; this is universally known as the Ziehl-Neelsen (ZN) technique. Despite being over 100 years old it remains a major tool for the rapid diagnosis of tuberculosis. Fluorescence microscopy has advantages where large number (Tiwari et al., 2003). 1.3. Sensitivity to physical and chemical agents Although Mycobacterium can survive for several weeks in the dark especially under moist conditions and for many days in dried sputum on clothing and in dust they are rapidly killed by ultraviolet light (including the component in daylight and sunlight) even through glass and by heat (60 °C for 15-20 minutes or by autoclaving). The tubercle bacilli are obligate pathogens but they survive in milk and in other organic materials and on pastureland so long as they are very sensitive (Grange, 2002). They are also heat sensitive and are destroyed in the process of pasteurization. Mycobacterium is susceptible to alcohol, formaldehyde and glutraldehyde and to a lesser extent to hypochlorites and phenolic disinfectants. They are considerably more resistant than other bacteria to acids, alkalis and ammonium compounds (Grange, 2002). M. tuberculosis was found to be more susceptible to acid pH and weak acids than M. smegmatis. The weak acids were more active against M. tuberculosis at acid pH than at neutral pH. M. tuberculosis was found to be less able to maintain its internal pH and membrane potential at acid pH than M. smegmatis. The antituberculous activity of weak acids correlated with their ability to disrupt the membrane potential but not the internal pH (Zhang et al., 2003). 5
  22. 22. Review of literature 1.4. Animal pathogenicity The success of M. tuberculosis as a pathogen is largely attributable to its ability to persist in host tissues. M. tuberculosis and M. bovis are both pathogenic to laboratory animals especially BALB/c mice (Aguilar et al., 2006) and the guinea pig (Laidlaw, 1989). Inoculate of as few as 10 bacilli can cause infection with death of the guinea pig in 6-15 weeks. Other Mycobacteria are less pathogenic for laboratory animals but mice are sometimes used for evaluation of new compound especially against M. avium infections (Gomez and McKinney, 2004). 1.5. Constituents of tubercle bacilli 1.5.1. Lipids: Mycobacterium is rich in lipids. These include mycolic acids (long chain fatty acids C78-C90) waxes and phosphates. In the cell the lipids are largely bound to proteins and polysaccharides. Lipids are to some extent responsible for acid fastness, removal of lipid with hot acids destroys acid fastness that depends on both the integrity of the cell wall and the presence of certain lipid. Acid fastness is also lost after sonication of the Mycobacteria cell. Analysis of lipids by gas chromatography reveals patterns that aids in classification of different species (Butler et al., 1991). 1.5.2. Proteins: Each type of Mycobacterium contains several proteins that elicit the tuberculin reaction. Proteins bound to wax fraction can upon injection induce tuberculin sensitivity. They can also elicit the formation of a variety of antibodies (Daffe and Etienne, 1999 and Hu et al., 2006). 1.5.3. Polysaccharides: Mycobacterium contains a variety of polysaccharides. Their role in the pathogenesis of disease is uncertain. They can induce the immediate type of hypersensitivity and can serve as antigen in reaction with sera of infected persons (Daffe and Etienne, 1999). 6
  23. 23. Review of literature 2. Tuberculosis Tuberculosis considered an important emerging disease in humans, is now the leading cause of death in adults worldwide (Cosivi et al., 1998; Kathleen et al., 2002 and Beck et al., 2005). TB is a disease of greatly antiquity tuberculous lesions have been found in the vertebrae of Neolithic man in Europe and of Egyptian mummies (Morse, et al., 1964 and Zink et al., 2001). TB has always been one of the great bacterial plagues. With the coming of the industrial revolution in Europe and the crowding of the population into cities the effects of the disease were dreadful by the mid 19th century. It was responsible for a third of all deaths in major cities such as Paris with the movement of explorers about the world the disease was also introduced into populations which had never before been exposed to it. The South Sea islanders suffered grievously with the disease at one time affecting more than 80% of all children. Even in the 17th century the poet John Bunyan aptly referred to the disease as the captain of all these men of death. In all populations it is particularly the overcrowded the malnourished and those with other diseases that are most susceptible (Mitchinson et al., 1996). 2.1. Epidemiology of tuberculosis M. tuberculosis infection remains the most successful human pathogen worldwide and more than one third of the world's population is exposed to the infection every year (Gagneux et al., 2006). Of this population more than 10 million develop clinical symptoms, and of those who remain untreated, perhaps 50 % will die (Dye et al., 1999 and Martin and Lazarus, 2000). The incidence of TB in different countries as estimated by the World Health Organization (WHO) vary from 23/100,000 and less in industrialized countries, 191/100,000 in Africa and 237/100,000 in South East Asia (Kart et al., 2003). The geographic distribution of TB has changed considerably over time. In the past, 7
  24. 24. Review of literature the highest levels of TB were found in the population of North America and northern Europe (Valerie et al., 2002). Today the highest annual risk of infection is encountered in the Andes, the Himalayas, sections of Indochina and the Philippines, Haiti and sub-Saharan Africa (Anane, 2003). Rates in North America and northern Europe are now low. According to a 1997 report from the Egyptian National TB Program, the annual risk of TB infection in this country is 0.32%. This report further revealed that the incidence of smear-positive cases in Egypt is 16 per 100,000 populations, with a rate of detection of new smearpositive cases of 70 % (Elmoghazy, 1997 and Said et al., 2001). Theistically distribution of TB, even in the develop world where the disease is uncommon, has always been predominantly at the lower socioeconomic level. TB is strikingly associated with poverty, particularly urban poverty. Because TB changes only slowly within a population, when that population moves from one place to anther it carries the risk of TB with it for the duration of the lifetime of the people who have moved (Valerie et al., 2002). Modern travel continues to be associated with risk of TB infection and disease TB transmission has been documented on commercial aircraft, from personnel or passengers to other personnel and passengers, but the risk of transmission is low. As in other settings, the likelihood of transmission is proportional to duration and proximity of contact. Travelers from low incidence to high incidence countries have an appreciable risk of acquiring TB infection similar to that of the general populations in the countries they visit, but the risk is higher if they work in health care (Al-Jahdali et al., 2003). 2.3. Risk factor The main risk factors for TB were marital status other than married, educational level less than higher, low income, having been in prison, not having own place of residence, current unemployment, current smoking (Liu et 8
  25. 25. Review of literature Figure 1. Epidemiology of TB in the world (WHO, 2000). 9
  26. 26. Review of literature al., 2004 and Altet-Gomez et al., 2005; Coker et al., 2006 and Davies et al., 2006), alcohol consumption, shortage of food, and contact with TB patients. Place of birth was not a risk factor. Risk of TB decreased for overweight persons (Tekkel et al., 2002). 2.3. Pathology of TB 2.3.1. Pulmonary TB Airborne tubercle bacilli produced by individuals with pulmonary TB and droplet nuclei remain suspended in the air for a long time (Wan et al., 2004). Once inhaled, fewer than 10% of M. tuberculosis organisms will reach the respiratory bronchioles and alveoli; most will settle in the upper respiratory epithelium, where they are likely to be expelled by the mucociliary escalator. Mycobacterium adheres specifically to extracellular matrix (ECM) which plays a role in the pathogenicity of Mycobacterium (Middleton et al., 2004). Bacteria that arrive in the deep lung are phagocytosed by alveolar macrophages and either killed or else survive to initiate an infection. Over the next 2 to 3 weeks, surviving organisms multiply and kill their host macrophages; this is followed by release of Mycobacterium and subsequent infection of additional host cells (Anand et al., 2006). The early exudate contains chemotactic factors that attract circulating monocytes, lymphocytes, and neutrophils, none of which kills the bacteria very efficiently. Enhanced production of monocytes and their early release from bone marrow can be observed clinically (Grange, 2002). Granulomatous focal lesions, composed of macrophage-derived epithelioid giant cells and lymphocytes, begin to form. Generally, the process of granuloma formation serves as an effective means for containing pathogens, preventing their continued growth and dissemination. Its success depends on 10
  27. 27. Review of literature both the number of macrophages at the site of infection and on the number of organisms present (Tsai et al., 2006). A host’s initial resistance to M. tuberculosis infection is directly proportional to the strength of this granulomatous response. TB granulomas display a relatively high rate of monocytes and lymphocyte turnover, attesting to the toxicity of the M. tuberculosis bacilli for the host cells, which must be continuously replaced by fresh recruits (Grange, 2002). While granuloma formation is quite an effective defense, even contained M .tuberculosis organisms are not always completely eradicated. Granuloma formation and destruction of Mycobacterium by macrophages are not antigen-specific events, and heat-killed or living M. tuberculosis bacilli are equally effective inducers of a granulomatous response (Grange, 2002). This observation contrasts with both delayed-type hypersensitivity (DTH) and cell-mediated immunity (CMI) (Jones-Lopez et al., 2006). In DTH, antigen-specific T-cell immune responses are evoked, and in CMI, live Mycobacterium is required for the development of protective immunity. In the first few days following infection, a strong granulomatous response is vital. However, after about 3 weeks, antigen-specific defenses develop and contribute greatly to the resolution of infection. With the emergence of a DTH response, infected macrophages in the interior of each granuloma are killed as the periphery becomes fibrotic and caseated (Matthew and Mary, 1996). After 4 to 5 weeks of progressive infection, microscopic granulomas enlarge, as individual foci expand and coalesce. This results in relatively large areas of necrotic debris, each surrounded by a layer of epithelioid histiocytes and multinucleated giant cells. These granulomas, or tubercles, are surrounded by a cellular zone of fibroblasts, lymphocytes, and blood-derived monocytes. Although M. tuberculosis bacilli are unable to multiply within this caseous tissue, due to its acidic pH, low availability of oxygen, and the presence of toxic fatty acids, some 11
  28. 28. Review of literature organisms may remain dormant there for decades. The strength of the host’s CMI responses determines whether an infection is arrested here or progresses to the next stages. With good CMI, the infection may be arrested permanently at this point. The granulomas subsequently heal, leaving small fibrous and calcified lesions (Houben et al., 2006). However, if CMI responses are insufficient, macrophages containing ingested but viable M. tuberculosis organisms may escape from the granuloma via the intrapulmonary lymphatic channels. This results in the rapid spread of the infection to the regional hilar lymph nodes. Where CMI is inadequate, the host’s DTH responses battle the ever-multiplying M. tuberculosis bacilli, but concomitantly, lung tissue is destroyed, leading to both pulmonary damage and the spread of organisms via the lymphatics and the blood to any organ of the body. As disease progresses further, the semisolid caseous center of the granuloma begin to soften and liquefy, providing a rich and oxygenated environment for extracellular Mycobacterial replication (Reinout et al., 2002). 2.3.2. Extra-pulmonary TB Extra-pulmonary tuberculosis is on the increase world over. Diagnosis of extra-pulmonary tuberculosis has always been a problem. It is a protean disease which can affect virtually all organs, not sparing even the relatively inaccessible sites. Extra-pulmonary tuberculosis can occur alone or in combination with the pulmonary variety. It is usually confined to a single site but disseminated form may also occur. Tuberculosis of meninges, spine, nervous system, abdomen, pleura, pericardium, bones and joints is considered of severe form compared to other sites (Engin and Balk, 2005 ). For a definitive diagnosis of tuberculosis, it is essential to culture the Mycobacterium. Many of the affected sites may require an invasive procedure to get a biological sample to reach a diagnosis (Lalit, 2004 and Khubnani and Munjal, 2005). 12
  29. 29. Review of literature Types of extra pulmonary TB Lymph node tuberculosis Lymph node TB is seldom complicated by systemic symptoms, except in people with HIV infection, in whom the bacterial load is large. The nodes are usually discrete, firm and nontender, but with time they may become fluctuant and drain spontaneously with sinus tract formation. Anterior and posterior triangles of the neck are the most common sites (in 70% of cases), followed by inguinal and axillaries sites. The best diagnostic procedure is excisional biopsy, which yields the diagnosis in 80% of cases. In the hands of a surgical expert, fine-needle aspiration biopsy is diagnostic in 60% of cases. A typical Mycobacterium lymphadenopathy is much more common than M. tuberculosis infection in children under the age of 5 years (Lee, 1995; Ashok et al., 2002; Narang et al., 2005 and Marais et al., 2006). Tuberculosis peritonitis Tuberculosis can involve any part of the gastrointestinal tract and is the sixth most frequent site of extrapulmonary involvement. Both the incidence and severity of abdominal tuberculosis are expected to increase with increasing incidence of HIV infection (Sharma and Bhatia, 2004).Tuberculosis bacteria reach the gastrointestinal tract via haematogenous spread, ingestion of infected sputum, or direct spread from infected contiguous lymph nodes and fallopian tubes. The gross pathology is characterized by transverse ulcers, fibrosis, thickening and stricturing of the bowel wall, enlarged and matted mesenteric lymph nodes, omental thickening, and peritoneal tubercles. Peritoneal tuberculosis occurs in three forms : wet type with ascitis, dry type with adhesions, and fibrotic type with omental thickening and loculated ascites 13
  30. 30. Review of literature (Humphries and Lam, 1998). The most common site of involvement of the gastrointestinal tuberculosis is the ileocaecal region. Ileocaecal and small bowel tuberculosis presents with a palpable mass in the right lower quadrant and/or complications of obstruction, perforation or malabsorption especially in the presence of stricture. Rare clinical presentations include dysphagia, odynophagia and a mid oesophageal ulcer due to oesophageal tuberculosis, dyspepsia and gastric outlet obstruction due to gastroduodenal tuberculosis, lower abdominal pain and haematochezia due to colonic tuberculosis, and annular rectal stricture and multiple perianal fistulae due to rectal and anal involvement. Chest X-rays show evidence of concomitant pulmonary lesions in less than 25 per cent of cases. Useful modalities for investigating a suspected case include small bowel barium meal, bariumenema, ultrasonography, computed tomographic scan and colonoscopy (Sharma and Bhatia, 2004). Ascitic fluid examination reveals straw coloured fluid with high protein, serum ascitis albumin gradient less than 1.1 g/dl, predominantly lymphocytic cells, and adenosine deaminase levels above 36 U/l. Laparoscopy is a very useful investigation in doubtful cases Chawla et al., (1986) reported that an optical density (OD) of 0.81 on ELISA and fluoroscent coefficient of 2.56 on soluble antigen fluorescent antibody as cut-off gave positivity of 92 and 83 per cent, respectively, with 12 and 8 per cent false positives respectively. Bhargava et al., (1992) used competitive ELISA with monoclonal antibody against 38 kDa protein and found a sensitivity of 81 per (Sharma and Bhatia, 2004). Genitourinary tuberculosis Genitourinary TB occurs with the hematogenous spread of tubercle bacilli to the glomeruli. The infection spreads in the genitourinary tract to involve renal pelvis, ureter, bladder, seminal vesicles, epididymis and testes. It is estimated that it takes between 8 and 22 years to produce a symptomatic renal lesion. Hence, it is a rare occurrence in children. The symptoms of genitourinary TB are 14
  31. 31. Review of literature those of bacterial pyelonephritis, recurring in spite of treatment; sterile pyuria is frequent. There may be concomitant pulmonary TB but not invariably. A solitary genital lesion may occur in men, but such a lesion is usually associated with urinary tract symptoms. The genital lesions in women are less often associated with renal disease but present with symptoms of chronic pelvic inflammation (Tzoanopoulos et al., 2003). The ratio of males to females was 66:39. The most common symptoms were flank pain, nocturia, frequent voiding and dysuria; testicular involvement was present in 16 % of cases. The definitive diagnosis depends on culture of the urine. During treatment it is important to follow the patient carefully to ensure potency of the lower collecting system. Urethral narrowing may require dilatation or stenting to prevent progressive obstruction (Makiyama et al., 2003; Ismail and Muhamad, 2003, Gibson et al., 2004 and Dam et al., 2006). Orthopedic tuberculosis Tuberculosis of the spine (Pott’s disease) is the most common site of bone infection in TB, accounting for 50% of cases. The large joints, the hip, knee, shoulder, elbow and wrist, are less common sites, and TB of the small joints is rare (Jutte and Van Loenhout-Rooyackers, 2006). Pott’s disease results from haematogenous spread of tuberculosis from other sites, often pulmonary. The infection then spreads from two adjacent vertebrae into the adjoining disc space. If only one vertebra is affected, the disc is normal, but if two are involved the intervertebral disc, which is avascular, cannot receive nutrients and collapses. The disc tissue dies and is broken down by caseation, leading to vertebral narrowing and eventually to vertebral collapse and spinal damage). A dry soft tissue mass often forms and super- infection is rare (Humphries and Lam, 1998). Tuberculous arthritis is an infection of the joints caused by tuberculosis. Approximately 1% of people affected with tuberculosis will 15
  32. 32. Review of literature develop associated arthritis. This form of arthritis can be very destructive to the tissues (Gülgün et al., 2000). The most common symptoms of skeletal TB are pain, tenderness and limitation of motion. About one-third of cases have softtissue fluctuance or sinus drainage. The accompanying systemic symptoms may include fever, weight loss and malaise. In most cases the blood count is normal, but the sedimentation rate is usually elevated (Gülgün et al., 2000). The diagnosis depends on biopsy for culture and pathologic examination of the affected tissues. The tissue surrounding the bony lesion shows granulomatous change, but the bacterial population is usually small, and culture results may be negative. Although radiographs are not diagnostic, computed tomography (CT) helps to target the biopsy site. Bone scans have been reported to give negative results in 35% of cases, and gallium scans in 70% of cases (Gülgün et al., 2000). Magnetic resonance of image is the modality of choice, because it can discriminate between abscess and granulation tissue and can delineate soft-tissue masses and identify the amount of bone destruction. In regions of the world where TB is common, it has been recommended in cases of suspected bone TB that treatment proceed without culture diagnosis because of the lack of appropriate facilities. However, in developed countries, where TB is less common, culture of a specimen before initiation of therapy is optimal, to confirm the diagnosis and to define the sensitivity of the organism. Surgery is recommended only for diagnostic biopsy, for patients with unstable or deformed spines, for those whose condition does not improve after 3 to 4 weeks of antibiotic therapy and for those in whom progressive neurological symptoms develop while they are receiving adequate treatment (Titov et al., 2004). Miliary tuberculosis Miliary TB refers to the tiny (less than 2 mm in diameter), discrete granulomatous lesions in lungs and other organs that result when blood-borne tubercle bacilli seed many tissues. The common sites include the spleen, the 16
  33. 33. Review of literature liver, the bone marrow, the kidneys and the adrenal glands, as well as the lungs, but any tissue may be involved. Hematogenously disseminated, M. tuberculosis infection may progress at the time of primary infection or years or decades later, at a time of immune suppression. The symptoms of miliary TB are fever, weight loss and weakness. Dyspnea suggests that the miliary lesion is causing hypoxemia. Findings of tachycardia, tachypnea, high temperature and splenomegaly are common. Choroid tubercles, small white lesions representing granuloma of the retina, are infrequent (Hussain et al., 2004). The findings on chest radiography, diffuse nodules of less than 2 mm diameter, are pathognomonic. In 40% of the patients, the results of chest radiography were reported as normal, but miliary lesions are often missed. Miliary TB may lead to adult respiratory distress syndrome. Hyponatremia is present in 10% of cases and may be due to inappropriate antidiuretic hormone secretion. Anemia occurs in two-thirds of cases of miliary TB and is usually the normochromic, normocytic form. Leucopenia may occur as a result of bone marrow infiltration with granuloma, but this is rare. Elevation of alkaline phosphatase level is not uncommon and usually reflects periportal granulomatous inflammation. The negative tuberculin skin test encountered in as many as 50% of cases of miliary TB should not dissuade the physician from making the diagnosis. When miliary TB is suspected and sputum examination does not reveal acid-fast bacilli, bone marrow or liver biopsy may lead to the correct diagnosis (Vasankari et al., 2003 and Donald et al., 2005). Tuberculosis of the central nervous system TB meningitis is the most common form of TB of the central nervous system (Sengoz, 2005 and Padayatchi et al., 2006), but solitary or multiple brain lesions, lesions of the spinal cord and even involvement of the ears and eyes have been reported. Classical tuberculous meningitis differs from acute bacterial meningitis in that it has a slower, more insidious onset. However, the 17
  34. 34. Review of literature symptoms are similar and include fever, anorexia, malaise, nausea, vomiting, headache and mental obtundation. The clinical symptoms have been described as presenting in 3 stages. Stage 1 has no neurologic signs, but there are symptoms of headache and fever. Stage 2 has focal neurologic abnormalities. People in stage 3, have the highest rates of mortality and neurologic sequelae (Castro et al., 1995; Whiteman, 1997; Gülgün et al., 2002 and Kulkarni et al., 2005). The pathogenesis of TB meningitis begins with the hematogenous seeding of the brain in a site adjacent to the meninges, which then ruptures into the subarachnoid space to produce meningitis. Only a small number of organisms are necessary to provoke the tissue reaction. A gelatinous exudate may collect at the base of the brain, interfering with cranial nerve function and provoking hydrocephaly. A vasculitic process is most commonly seen at the base of the brain and may cause infarction and neurologic sequelae (Gülgün et al., 2002). Diagnosis of tuberculous meningitis depends on a high index of suspicion, especially in children, and recent contact with a case of TB. In cases of TB meningitis, the cerebrospinal fluid initially shows leukocytosis, but over a period of days, the predominant cell is the lymphocyte. The protein level is elevated and the glucose level decreased. The cerebrospinal fluid is seldom positive on direct smear examination for acid-fast bacilli (in only 25% of cases), but the proteinaceous pellicle may capture organisms and should be removed from standing cerebrospinal fluid and stained. It may take 10 days to 8 weeks for positive culture results to appear. Therefore, antituberculous drug treatment should be started immediately, while awaiting the results. Any delay in the institution of treatment increases the risk of progressive neurologic sequelae (Kashyap et al., 2003; Youssef et al., 2004 and Donald et al., 2005). 18
  35. 35. Review of literature Tuberculosis of sinusitis: TB sinusitis is a rare occurrence, the diagnosis of TB sinusitis is usually based on: (1) the absence of clinical response to usual antibiotics (2) the presence of caseous granulomatous inflammatory lesion on histopathology, and (3) identification of Mycobacterium tuberculosis by bacteriological culture or polymerase chain reaction assay. Antineutrophil cytoplasmic antibody helps differentiate Wegener’s granulomatosis, although this test is negative in 15% of localized disease (Beltran et al., 2003). Other sites TB may reactivate at any site of hematogenous dissemination. TB pericarditis and TB of the eye, ear, skin or soft tissue are infrequent (Shimada et al., 2003; Fenniche et al., 2003; Crum, 2003 Bulbuloglu et al., 2006 and Nalini and Vinayak, 2006). 2.4. Control of tuberculosis TB is preventable by the early detection of patients with active TB and careful follow up of their contacts with tuberculin tests, X-rays and appropriate treatment and vaccination are the main stays of public health TB control (Grange, 2002; Kim et al., 2003; Mitchison, 2005 and Brassard et al., 2006). The history of chemotherapy of TB commenced in 1944 with the discovery of streptomycin. Currently, short-course chemotherapy comprising rifampicin, isoniazid, pyrazinamide and ethambutol /streptomycin administered under directly observed settings for 6 months (initially all four drugs followed by the former two drugs), constitutes the cornerstone treatment for pulmonary TB (Davies and Yew, 2003 and Theobald et al., 2006). BCG (Bacillus CalmetteGuerin) vaccine was developed from an attenuated strain of M. bovis at the beginning of the twentieth century. Its widespread use as a vaccine against 19
  36. 36. Review of literature tuberculosis. The vaccine was originally given orally to neonates but it is now given by intradermal injection. It remains one of the most frequently administered vaccines in the world. It has also been one of the most controversial. Widely differing estimates of the effectiveness of BCG at protecting against different forms of tuberculosis in different population subgroups in different settings have been published. Some countries, with a low incidence of tuberculosis, did not adopt the use of BCG vaccine at all and some others abandoned its use at a later stage. In addition, great variation developed in national programmes for the administration of BCG including the age at which it should be given, whether or not its administration should be preceded by tuberculin sensitivity testing, and whether repeat vaccinations with BCG should be given (Grange, 2002). In recent decades, some consensus has been reached about the role of BCG vaccination in populations where it appears to offer some protection. Protection appears to be greatest in infants and children and against the early primary progressive forms of disease (including disseminated disease and meningitis). Protection against disease resulting from secondary reactivation, particularly pulmonary disease in adults, appears to be much more limited. As this is the group of cases responsible for most transmission of infection, BCG vaccination probably has very limited impact on controlling the incidence of new infections in the community. In addition, the evidence that repeat vaccination offers additional protection is very limited (Gradmann, 2006). Identification of complete BCG genome in 1998 has opened new vistas in newer BCG vaccine development (Rahaman et al., 2001; Parthasarathy, 2003; Wedlock et al., 2005; Haile and Kallenius, 2005; Collins et al., 2005; Langermans et al., 2005; Williams et al., 2005 and Dietrich et al., 2006). 20
  37. 37. Review of literature 3. Diagnosis of tuberculosis Despite the efforts for control and eradication of TB, new cases of the disease are diagnosed daily. The diagnosis of TB is easily made when the classical features of pulmonary necrotizing granulomatous inflammation are seen. However, extra-pulmonary lesions may clinically and radiographically mimic a neoplastic process, and this may lead to misdiagnosis and delay in treatment (Hwang et al., 2004; Beck et al., 2005 and Nahid et al., 2006). Rapid and accurate diagnosis of symptomatic patients is a cornerstone of global TB control strategies. Remarkable progress has recently been made upgrading the speed and quality of mycobacteriology diagnostic services in developed countries, but for most of the world where TB is a large public health burden those gains are still unrealized. The design and quality of clinical trials evaluating new diagnostics must be improved, clinical and laboratory services that would allow rapid response to test results need to be enhanced, and basic and operational research to appraise the impact and cost-effectiveness of new diagnostic technologies must be carried out (Mark and Perkins, 2000). 3.1. Microscopic method 3.1.1. Ziehl - Neelsen staining technique The diagnosis of Mycobacterial infection depends on the Ziehl-Neelsen (ZN) stain, which detects Mycobacterium because of their characteristic acidfast cell wall composition and structure (Nahid et al., 2006). The histological diagnosis of tuberculosis (TB) comprises various aspects: (1) sensitive detection of Mycobacterium; (2) precise localization of Mycobacterium in the context of granulomatous lesions; (3) staging of disease according to Mycobacterial spread and granulomatous tissue integrity. Thus, detection of minute numbers of acidfast bacteria in tissue specimens is critical (Bishop and Neumann, 1970). In 1882, Ehrlich discovered that Mycobacterium with fuchsin (in the presence of 21
  38. 38. Review of literature aniline oil as mordant) resist decolorization by mineral acids. In the same year Ziehl changed the mordant to carbolic acid and in 1883 Neelsen increased the concentration of carbolic acid incorporated it with the dye to form carbol fuchsin thus the standard stain for demonstrating acid-fastness was formulated the ZN stain (Bishop and Neumann, 1970). Many modifications have been described but all basically use carbolfuchsin to stain the organisms and mineral acids to decolorize the background. The background is then counterstained with another dye such as malachite green or methylene blue to give red acid - fast bacilli against a green or blue background as shown in figure 2. Some methods also use alcohol for decolorization this give a cleaner slide but it is important to realize that not all Mycobacterium. In the ZN staining technique heat fixed smears of the specimen are folded with a solution of carbol fuchsin (a mixture of basic fuchsin and phenol) and heated until steam rise. After washing with water, the slide is flooded with a dilute mineral acid (e.g. 3 % hydrochloric acid) and after further washing a green or blue counterstian background color seen (Chessbrough, 2000). Concentration of acid-fast bacilli (AFB) in clinical specimens is an important step in the laboratory diagnosis of mycobacterial diseases. Microscopy of smears of sputum by direct and after mechanical sedimentation and centrifugation methods followed by treatment with 5% sodium hypochlorite (NaOCl) solution for concentration of the organisms were compared and evaluated. The rate of recovery of AFB from sputum was 8.5%, 25.5% and 38.0% for direct smear microscopy, concentration by sedimentation of NaOCltreated sputa followed by ZN staining and concentration by centrifugation after use of NaOCl respectively. Both the concentration methods by the use of NaOCl solution increased the yield of the AFB by more than and centrifugation by the treatment of NaOCl increased the sensitivity to 75% and 77.9% respectively, 22
  39. 39. Review of literature Figure 2. Acid fast bacilli (shown in red) are tubercle bacilli (Grange, 2002) 23
  40. 40. Review of literature and the specificity to 100% for both techniques (Gebre-Selassie, 2003; Buijtels and Petit, 2005). Kim et al., (2003) developed an automated stainer for AFB and evaluated its usefulness in comparison with manual staining. The key feature of automated stainer is a heating apparatus required for fixation and carbol-fuchsin staining. After smear slides are placed into the machine, the entire staining process is fully automated, from fixation to final washing and drying. With the automated methods, five slides can be fixed and stained in 21 min at consistent high quality. Using sputum samples from 91 TB patients, the staining results of the automated stainer were compared blindly with those of manual staining. The concordance rate between the two methods was 94.5%. 3.1.2. Auramine phenol fluorochrome staining technique: The auramine phenol fluorochrome staining technique is used to detect M. tuberculosis in sputum, cerebrospinal fluid and other specimens. It is recommended in preference to the ZN technique because a large area of a smear can be examined which increases the possibility of detecting the tubercle bacilli due to enable a much greater area of the smear to be examined in shorter time. Auramine is a fluorochrome, that is, dyes that will fluoresce when illuminated (excited) by blue violet or ultraviolet (UV) light. No heating of the stain is required. After being stained with auramine the smear is decolorized with an acid alcohol solution which removes the dye from the background. The auramine is not removed from the tubercle bacilli. After being decolorized the smear is washed with a weak solution of potassium permanganate to darken the background. Tubercle bacilli fluoresce white-yellow background (Chessbrough, 2000 and Murray et al., 2003). 24 against the dark
  41. 41. Review of literature Biopsies Granulomas of varying size, predominantly consisting of aggregated epithelioid macrophages, were found in most of the organs and tissues examined and were consistently present in the liver, spleen, lymph nodes, and lungs. Such granulomas were occasionally noted in the adrenal gland, kidney, myocardium, pancreas, epididymus, pleura, intestine, peritoneum, and skin. Lesions were absent from the brain, skeletal muscle, urinary bladder, and testis. The smaller granulomas consisted purely of macrophages, while large ones showed central necrosis and sometimes contained small aggregates of lymphocytes and plasma cells. Giant cells were rare, and no calcification was seen. Acid-fast rods, typical of Mycobacterium species, were noted in the cytoplasm of macrophages in all eight mongooses but varied in numbers from scarce to abundant (Kathleen, 2002). 3.2. X - ray Methods for the radiographic diagnosis of tuberculosis have improved from simple fluoroscopy to computerized tomography (Mitchison, 2005 and Nahid et al., 2006). Evidence of pulmonary TB in chest radiographs varies but usually radiographs show enlargement of hilar, mediastinal, or subcarinal lymph nodes and lung parenchymal changes. Most of the radiographic abnormalities are caused by a combination of lung disease and the mechanical changes induced by partial or complete airway obstruction resulting from enlarging intrathoracic nodes. The most common findings are segmental hyperinflation then atelectasis, alveolar consolidation, interstitial densities, pleural effusion, and, rarely, a focal mass. Cavitation is rare in young children but is more common in adolescents, who may develop reactivation disease similar to that seen in adults (Gülgün et al., 2002). Parenchymal-infiltrate lesions are the most frequent radiological manifestation of pulmonary TB, and they are generally associated with cavities and there is a relationship between the presence of acid 25
  42. 42. Review of literature fast bacilli in sputum and pulmonary cavity lesions (Gomes et al., 2003). The development of radiographic techniques, such as computed tomography (CT) scanning may show enlarged or prominent mediastinal or hilar lymph nodes in some children with recent TB infection and a normal chest radiograph (Parisi et al., 1994). In the absence of a CT scan, the child's disease stage would be called TB infection, and single drug therapy would be used. CT scan can be helpful in selected cases to demonstrate endobronchial disease, pericardial invasion, early cavitation, and bronchiectasis resulting from pulmonary TB when the chest radiograph is abnormal but the pathologic process is not clear (Delacourt et al., 1993 and Gülgün et al., 2002). 3.3. Culture Tubercle bacilli are able to grow on a wide range of enriched culture media but Lowenstein Jensen (LJ) medium is the most widely used in clinical practice. This consists of whole eggs, glycerol, asparagine and some mineral salts and is solidified by heating (inspissation). Malachite green dye is added to the medium to inhibit the growth of some contaminating bacteria and to provide a contrasting color against which colonies of Mycobacteria are easily seen. Agar-based media or broth’s enriched with bovine serum albumin are also used. produce visible growth on LJ medium in about 2 weeks although on primary isolation from clinical material colonies may take up to 8 weeks to appear. Colonies are buff color and often have a dry breadcrumb like appearance (Grange, 2002). Growth is characteristically heaped up and luxuriant or eugenic in contrast to the small flat dysgenic colonies of bovine tubercle bacilli on this medium. The growth of Mycobacterium is much better on media containing Sodium pyruvate in place of glycerol e.g. Stonebrink’s medium. Tubercle bacilli have a rather limited temperature range of growth, their optimal growth temperature is 35-37 °C but they fail to grow at 25 or 41°C. Most other 26
  43. 43. Review of literature Mycobacteria grow at one or other or both of there temperature. Like all Mycobacteria the tubercle bacilli are obligate aerobes but Mycobacterium grows better in conditions of reduced oxygen tension. Thus when incorporated in soft agar media M. tuberculosis grows on the surface while Mycobacterium bovis grows as a band a few millimeters below the surface. This provides a useful differentiating test (Grange, 2002). In recognition of their superior speed and sensitivity, radiometric liquid culture systems have been in common use in level III mycobacteriology laboratories in developed countries for more than a decade. The difficulty working with radioactive materials, the necessity of expensive apparatus for the detection of radioactive gas and the cost of materials limit the use of these systems. Recently, alternative growth detection methods for liquid culture employing oxygen quenching and redox reagents have been described and commercialized that show performance comparable to BACTEC 460 tuberculosis (Liu et al., 1999; Cambau et al., 1999 ; Somoskِ i and Magyar, v 1999; Hanna et al., 1999 and Heifets et al., 2000). Though these methods offer an attractive enhancement (not replacement) to culture on Lِ ensteinw Jensen or other solid media, the cost of these commercial systems is currently considered too high. For susceptibility testing, several of these growth detection methods for liquid culture have demonstrated comparable performance to standard methods (Caviedes et al., 2000; Baylan, 2005 and Al-Hakeem et al., 2005). 3.4. Tuberculin skin testing It is an allergic skin test used in diagnosis of TB infection, it is mediated by specifically sensitized small T-lymphocytes which interact with Mycobacterium antigen with the release of acute factors called lymphokines results in typical cellular reaction (Comstock, et al., 1981). Robert Kock first demonstrated tuberculin hypersensitivity in 1891(Gradmann, 2006). During his 27
  44. 44. Review of literature experiments with TB he showed that guinea pigs infected with M. tuberculosis within two or more weeks earlier reacted differently from uninfected ones to the subcutaneous re-injection of virulent living tubercle bacilli where at the site of inoculation a massive inflammatory reaction developed within two days. Extension to regional lymph nodes either delayed or did not occur; the normal animals infected with similar material developed progressive TB. He also demonstrated that these changes could be produced by dead as well as living microorganism. Bacteria free protein fraction extract prepared from these organisms known as Kock old tuberculin. Autoclaving and filtering autolyzed 8 week prepared the latter reagent used in skin testing old liquid cultures of M. tuberculosis. The protein content of this preparation purified first with tricholoroacetic acid and later with ammonium sulfate was termed purified protein derivative (PPD). This amount of tuberculin has been chosen as one that gives maximal sensitivity with minimal adverse reactions (Brooks et al., 1998). 3.4.1. Dose of Tuberculin A large amount of tuberculin injected into a hypersensitive host may give rise to severe local reactions and a flare up of inflammation and necrosis at the main sites of infection (focal reactions). For these reason tuberculin tests in survey employ 5 Tu in persons suspected of extreme hypersensitivity, skin testing is begun with 1 Tu. More concentrated (250 Tu) is administered only if the reaction to 5 Tu is negative. The volume is usually 0.1 ml injected intragermally (Grange, 2002). 3.4.2. Reactions to Tuberculin In an individual who has not had contact with Mycobacterium there is no reaction to PPD. An individual who has had a primary infection with tubercle bacilli develops induration, edema and erythema in 24-48 hours and with very intense reaction even central necrosis. The skin test should be read in 48 or 72 hours. It is considered positive if induration 10 mm or more in diameter follows the injection of 5 Tu. Positive test tends to persist for several days. Weak 28
  45. 45. Review of literature reactions may disappear more rapidly. The tuberculin test becomes positive within 4-6 weeks after infection (or injection of virulent bacilli). It may be negative in the presence of tubercle infection when allergy develops due to miliary TB, measles, Hodgkin’s disease, sarcoidosis or AIDS (Flament and Perronne, 1997 and Grange, 2002). To overcome the poor specificity of the existing skin test based on tuberculin, newer tests with defined antigens are needed to discriminate between the infected individuals from those with active disease. The latest of these is the MPB 64. MPB 64 is a specific mycobacterial antigen for M .tuberculosis complex. This patch test becomes positive in 3-4 days after patch application and lasts for a week. The test has a specificity of 100% and a sensitivity of 98.1% (Nakamura, 1998). Another approach is the use of defined antigens for an accurate and rapid test for tuberculosis infection based on the detection of T cells sensitized to M. tuberculosis either by blood tests in vitro or skin tests in vivo (Anderson et al., 2000). Mononuclear cells from the peripheral blood are stimulated in vitro and production of interferon gamma from the sensitized T cells is measured by ELISA33. The antigens used are ESAT 6 (early secretory antigen TB) and CFP 10 (culture filtrate protein), which are being used as alternatives for PPD, for use in skin test (tuberculin testing) in vivo (Brock et al., 2001). 3.5 Adenosine deaminase activity (ADA) ADA, an enzyme that catalyzes the deamination of adenosine and deoxyadenosine into inosine and deoxyinosine, is found in most cells (Valdes et al., 1996). ADA analysis is a simple and inexpensive colorimetric test that can be performed on body fluids (Valdes et al., 1993; Mishra et al., 2000; Sharma and Banga, 2005 and Reuter et al., 2006). Several studies have suggested that an elevated pleural fluid ADA level predicts tuberculous pleuritis with a sensitivity of 90-100% and a specificity of 89-100% when the Giusti 29
  46. 46. Review of literature method is used. The reported cutoff value for ADA (total) varies from 47 to 60 U/L (Valdes et al., 1995; Burgess et al. 1995 and Villena et al., 1996). Using a cut off value of total CSF adenosine deaminase activity of >6 U/L, in one study on 11 patients with TB meningitis, 9 with cryptococcal meningitis, 13 with acute bacterial meningitis and 9 with aseptic meningitis, the sensitivity of total ADA for detecting TB meningitis was 90.9% and specificity was 94% in all patients and 77.3% compared with those with cryptococcal meningitis or acute bacterial meningitis. Other studies have shown sensitivities of 44-100% and specificities of 75-99% for total ADA (Petterson et al., 1992; Gambbir et al., 1999 and Eintracht et al., 2000). Ascetic fluid ADA activity has been proposed as a useful diagnostic test for diagnosis of TB peritonitis. Six of seven studies outside the united states have reported 100% sensitivity for the diagnosis of peritoneal TB, with specificities in the range of 92-100% (Fernandez et al., 1991 and Balian et al., 2000). 3.6. Serodiagnosis of TB The diagnosis of TB is based primarily on the identification of Mycobacterium by clinical and radiographic evidence. However bacteriological examination is frequently times consuming while radioscopy or fluorography is not always available. The detection of antigens and antibodies has been widely used in attempts to diagnose TB since the end of the 19th century. In the 20th century periods of high optimism have alternated with periods of total pessimism with regard to the role of serological tests in TB diagnosis (Khomenko et al., 1996). Two principal components are necessary for successful serodiagnosis a technically simple and reproducible test and highly specific reagents i.e. antigens to detect circulating antibodies (Fujita et al., 2006) and antibodies to detect antigens. In recent decades modification of a number of tests employing automated recording of results and requiring small amounts of blood have been developed, such as radioimmunoassay (RIA) and 30
  47. 47. Review of literature enzyme-linked immunosorbent assays (ELISA). RIA and ELISA have been employed in serodiagnosis of TB (Daniel et al., 1999; Chan et al., 2000 and Mark and Perkins, 2000). Antibodies to mycobacterial antigens in sera of patients are detected either by using monoclonal or polyclonal antibodies. Crossreaction by environmental Mycobacterium is likely to produce false positive results. Reproducible methods for purification of mycobacterial antigens have yet to be evolved; hence the results of most assays available at present are variable in different settings. Some of the newer approaches are as follows (Ramachandran and Paramasivan, 2003). Immunochromatographic test: TB STAT-PAK It is based on the detection of antibodies and it has been evolved with a capability to differentiate between active or dormant TB infection in whole blood, plasma or serum. Its value in disease endemic countries such as India is yet to be ascertained (Bathamley, 1995). Enzyme immuno- assay Superoxide dismutase is an important secretory protein of M. tuberculosis and has been evaluated for the serodiagnosis of tuberculosis. It is found to be useful only in low prevalence countries (93-94% positive predictive value), compared to high prevalence countries like India and Egypt, where the positive predictive value drops to 77-88% (Ramachandran and Paramasivan, 2003). Insta test TB It is a rapid in vitro assay for the detection of antibody in active TB disease using whole blood or serum. The test employs an antibody binding protein conjugated to a colloidal gold particle and a unique combination of TB antigens immobilized on the membrane (Chan et al., 2000). Some of the other commercially available antibody tests for pulmonary TB are listed in table 1. 31
  48. 48. Review of literature Table 1. Some antibody tests for diagnosis of pulmonary tuberculosis (Ramachandran and Paramasivan, 2003). Name of the assays Antigen used MycoDot (Dot-blot) Lipo arabinomannan (LAM) Detect-TB (ELISA) Recombinant protein Peptide Pathozyme Myco (ELISA) 38 kDa (recombinant Ag) and LAM Pathozyme TB (ELISA) 38 kDa (recombinant) Antigen A60 (ELISA) Antigen 60 ICT diagnostics (membrane based) 38 kDa (recombinant) 32
  49. 49. Review of literature 3.7. Polymerase chain reaction (PCR) PCR allows sequences of DNA present in only a few copies of Mycobacterium to be amplified in vitro such that the amount of amplified DNA can be visualized and identified. If appropriate sequences specific for M.tuberculosis are selected, 10-1000 organisms can be readily identified. The PCR methodology is rapid; results are available within a day of DNA extraction from the sample. A number of target genes of mycobacterial DNA have been evaluated for diagnosis by PCR and various other genotypic methods (Pfyffer, 1999). The most common target used in the PCR is IS6110. This sequence is specific for M. tuberculosis complex and is present up to 20 times in the genome, thus offering multiple targets for amplification. PCR detection of IS6110 in sputum (in pulmonary TB) and peripheral blood (in extra-pulmonary TB), when compared to culture has a sensitivity, specificity and positive predictability of 83.5, 99.0 and 94.2% respectively. A variety of PCR methods have been described in the search for a sensitive and reliable screening test for tuberculosis in clinical specimens. Species-specific and genus specific PCR methods are being used with various targets and modifications of PCR. The following are some of the methods used for identification of M. tuberculosis and non M. tuberculosis (Shaw and Taylor, 1998 and Grassi, et al., 2006). 3.7. 1. Nucleic acid amplification (NAA): This approach identifies the presence of genetic information unique to M. tuberculosis complex directly from pre-processed clinical specimens (Chedore et al., 2006). The NAA technique uses chemical, rather than biological amplification to produce nucleic acid, so that within a few hours these tests distinguish between M. tuberculosis complex and non M. tuberculosis in an AFB positive specimen. It is currently used only for respiratory specimens; use for non-respiratory specimens is likely in the near future. A positive direct 33
  50. 50. Review of literature amplified test in conjunction with an AFB-positive smear is highly predictive of tubercular disease. However, the results of NAA are preliminary; mycobacterial culture is still needed for species identification/confirmation and for drugsusceptibility testing. A negative NAA with an AFB-positive smear indicates that the AFB is probably non M. tuberculosis. However, there are occasional false-negative or false positive results being reported, which are either due to the presence of fewer bacilli or due to contamination. Another disadvantage of the technique is that both viable and dead bacilli can give positive results as the DNA of both can be amplified (Ramachandran and Paramasivan, 2003). 3.7. 2. Ligase chain reaction: It is a variant of PCR, in which pair of oligonucleotides are made to bind to one of the DNA target strands, so that they are adjacent to each other. A second pair of oligonucleotides is designed to hybridize to the same regions on the complementary DNA. The action of DNA polymerase and ligase in the presence of nucleotides results in the gap between adjacent primers being filled with the appropriate nucleotides and ligation of the primers. The LCX M. tuberculosis assay is mainly being used for respiratory samples, and has a high overall specificity and sensitivity for smear positive and negative specimens (Ramachandran and Paramasivan, 2003). 3.8. Mycobacterial antigen detection: The advent of nucleic acid amplification technology (especially PCR) has overshadowed recent developments in antigen detection. However, free mycobacterial antigen at a concentration of 3-20 ng/ml can be detected in biological fluids such as pleural fluid or cerebrospinal fluid (Mathai et al., 2003). Most of the tests use polyclonal antibodies raised against crude mycobacterial antigens except for antigen 5 and lipoarabinomannan (LAM). The sensitivity of tests ranges from 40-50% and specificity 80-95%. The methods 34
  51. 51. Review of literature used for antigen detection are: the sandwich ELISA, inhibition ELISA, latex agglutination and reverse passive haemagglutination tests (Arais et al., 2000). Antigen detection has been evaluated in sputum (sensitivity 60%, specificity 91%) (Ramachandran and Paramasivan, 2003), pleural fluid (80% and 38%), bronchoalveloar fluid (67% and 85%) and serum (45% and 100 %). The generally poor results reflecting the difficulty of detecting antigen in very cellular samples. However, studies on CSF (sensitivity 75%, specificity 98%) have been encouraging. Mycobacterial antigen detection has been evaluated in clinical samples from adults (Radhakrishnan et al., 1990). A quantitative test to detect LAM has been developed for the detection of TB in urine specimens. Another test being used in a field trial is the dipstick method (semi-quantitative) for the detection of LAM in both pulmonary and extra-pulmonary specimens. Preliminary reports have shown a sensitivity and specificity of 93 and 95% respectively (Del Prete et al., 1998 and Ramachandran and Paramasivan, 2003). 3.8.1. Application of monoclonal antibodies in diagnosis of M. tuberculosis antigen The monoclonal antibody is an antibody preparation in which all the molecules are identical and have precisely the same variable and constant amino acid sequences in both heavy and light chains. Monoclonal antibody is an antibody synthesized by a single clone of B lymphocytes or plasma cells. The first to be observed were produced by malignant plasma cells in patients with multiple myeloma and associated gammopathies. The identical copies of the antibody molecules produced contain only one class of heavy chain and one type of light chain (Julius and Robert, 2000). The first report of hybridoma production was in fact in early 1970s with virus specific lymphocytes together with tumor cells and subsequent reports of both inter species and human hybridoma appeared in the literature before the full 35
  52. 52. Review of literature potential of the technology was expanded by (Kohler and Milstein, 1975). The hybridization technique was based on fusion between splenocytes of a mouse immunized with a particular antigen and myeloma cells. The resulting hybrid cells express both the lymphocytes property of specific antibody production and the immortal character of the myeloma cells which is necessary to grow the produced hybrid to survive. The mixture of fused and unfused cells was placed into multiple small tissue culture wells in a specific selection medium called HAT. HAT medium contains hypoxanthin aminopterin and thymidine and used to select out the fused cells only. Through this medium spleen cell will die after short times of culture and myeloma cells will die because it can not use hypoxanthin and thymidine that present in HAT medium due to lack of hypoxanthin guanine phosphoribosyl transferase (HPRT) enzyme thus the fused cells (hybridoma cells) are continue to grow in culture. The hybrid cells is isolated and allows growing into a homogenous colony of cells (cloning process), this colony can be selected and grown to provide the secreted monoclonal antibody (Julius and Robert, 2000). The cloned hybrid cells are then injected into mice to form ascetic producing tumors thereby increasing the antibody concentration to 1000 fold. Hybridoma will expand in the peritoneal cavity of animal of the same strain as the tumor cell line donor and spleen cell donor and secrete monoclonal antibody into the ascetic fluid formed within the cavity. By this produced large amounts of a monoclonal antibody can be produced (10-60 mg/ml) without the need for large-scale cell culture (Julius and Robert, 2000). Attempts have been directed towards identifying mycobacterial antigens in biological fluids by employing polyclonal and monoclonal antibodies specific for M. tuberculosis (Chernousova et al.,1995 and Kumar et al., 2000). Cho et al., (1992) produced a monoclonal antibody (MAbIII604) specific to phenolic glycolipid TB (PGL-TB), a M. tuberculosis-specific antigen, and 36
  53. 53. Review of literature used in the detection of the antigen. MAbIII604 reacted with the PGL-TB antigen but not with other phenolic glycolipids from M. leprae, M. bovis and M. kansasii thus indicating the specificity of the monoclonal antibody to PGL-TB. Cummings et al., (1996) produced murine monoclonal antibodies against M. tuberculosis, one monoclonal antibody HB28, demonstrated high level specific reactivity to M. tuberculosis. Western blot analysis demonstrated reactivity to a single 65-kDa M. tuberculosis protein in the cell wall extract and culture filtrate. Avdienko et al., (1996) produced monoclonal antibodies (mAb) against M. tuberculosis H37Rv. The mAb acted against M. tuberculosis H36Rv with molecular mass 14, 17-15, 25 27 30 kDa excluding mAb S5B3B8 and S3H5D7 which acted against the main antigen with 54 kDa mass and 5-6 bands of antigens. Kumar et al., (2000) produced ten mAb designated TRC 1-10 were produced against M. tuberculosis H37Rv culture filtrate were raised by immunizing BALB/c mice and characterization. Of these, 7 mAb, TRC 1-7 reacted with the 30/31 kDa doublet (antigen 85 complex), TRC 8 with 12 kDa in addition to 30/31 kDa and TRC 9 and 10 with the 24 and 12 kDa antigens respectively. 37
  54. 54. Materials and Methods Materials and Methods 1. Samples:1.1. Serum samples Serum samples of 506 individuals (383 males, 123 females; aged 14-58 year) were obtained from the Department of Chest Diseases at Sayd Galal University Hospital, Al-Azhar University, Cairo, Egypt. Blood samples were allowed to clot for separation of sera. Tubes were centrifuged at 4000 rpm for 10 minutes serum were separated and stored at – 20 ºC. Patients with pulmonary TB (n = 296) were diagnosed by sputum smear for acid-fast bacilli or by culture for M. tuberculosis and all had no prior clinical history of TB. Patients with extra-pulmonary TB (n = 93) were diagnosed by clinical symptoms, radiographic evidence, and ultrasound, or a combination of these techniques, depending on the location of the infection in each patient. The sites of extrapulmonary tuberculosis were peritonitis (n = 25), meningitis (n = 22), lymphadenitis (n = 14), genitourinary tract (n = 19), potts disease (n = 5), arthritis (n = 3), sinusitis (n =3), millary (n = 2). None had clinical or radiological evidence of concurrent active pulmonary tuberculosis. In addition, sera of patients admitted to the hospital for a defined acute or chronic non-tuberculous diseases (n=69) including; chronic obstructive pulmonary disease (n =30), asthma (n =10), ischemic heart disease (n =10), pneumonia (n =5), bronchitis (n =5), lung cancer (n =5) and lung infection (n =4) as well as sera of n = 48 healthy volunteers with no signs of clinical impairment and normal chest radiographs were included as controls. 1.2. Cerebrospinal fluid (CSF) of tuberculous meningitis: CSF samples were obtained from 22 patients with tuberculous meningitis (15 males, 7 females; aged 36-50) and before antibiotic therapy. They were considered likely to have meningitis on the basis on clinical features, such as 38
  55. 55. Materials and Methods neck rigidity, positive Kernig's sign and compatible CSF biochemical parameters, viz., elevated protein levels (60-400 mg% mean 98 mg%), low glucose concentration (8-30 mg% mean 23 mg%) and pleocytosis (30-700 cells/cm3) in their CSF specimens. Patients who had received intravenous antibiotic were excluded from our analysis. These patients had neither manifestations of pulmonary tuberculosis nor had received chemotherapy for tuberculosis in the recent past. The CSF specimens were collected from all patients under aseptic conditions and were centrifuged at 5000 x g for 30 min. The deposits were examined by Ziehl-Neelsen staining. 1.3. Tuberculous ascetic fluid from tuberculous peritonitis: Ascites fluid specimens were obtained from 25 patients with tuberculous peritonitis (20 males, 5 females; mean age 45-58) and centrifuged at 4,000 r.p.m for 10 min. The deposits were examined microscopically for acid-fast bacilli (Ziehl-Neelsen staining). The supernatants were coded and used for the detection of antigen. Five patients with non-tuberculous ascites (4 transudative and 1 exudative) negative for M. tuberculosis by smear were used as controls. 1.4. Bacilli Calmette-Guerin (BCG) as source of M. tuberculosis: BCG was as purchased from Egyptian organization for biological products and vaccines (Giza, Egypt). The protein content of BCG was 5 mg/ml. 2. TB-55 Monoclonal antibody: An IgG anti–M. tuberculosis mouse monoclonal antibody, was prepared using hybridoma technique (Attallah et al., 2003). In brief, M. tuberculosis was grown at 37º C for 4-6 weeks on Lowenstein – Jensen medium. Total bacterial culture filtrate was collected by filtration through 0.45-µm cellulose acetate membranes, and then dialysed at 4º C against 0.01 M PBS, pH7.2, for 24 h. The 39
  56. 56. Materials and Methods dialyzed filtrate was stored at -70º C for 60 min, lyophilized, and reconstituted With 0.01 M PBS, pH 7.2. Proteins content was determined and it was stored at -20ºC. Balb/c female mice were intraperitonealy immunized using the dialyzed bacterial culture filtrate. Spleen cells were taken from immunized mice and fused with P3-X63-Ag8-UI mouse myeloma cells. The resulting hybrids were tested for the presence of specific antibodies against M. tuberculosis cultural filtrate using an indirect enzyme linked immunosorbent assay (ELISA). The highly positive hybrids were cloned by the limiting dilution method. One of the highly reactive cell lines (designated TB-55) indicating the specificity of the developed TB-55 mAb to M. tuberculosis was injected intraperitonealy into Balb/c mice for ascites production. The ascites were collected, centrifuged to remove the debris, and stored at -20º C until used 3. Protein content determination: The protein content of the antigenic solutions (BCG, TB-55 mAb, ascitic fluid, CSF and diluted serum samples 1:100) were measured colorimetrically using the method of Lowry, et al. (1951). The colorimetric quantitation of protein by use of the Folin - Ciocalteu reagent depends on the tryptophan contents of the protein. The intensity of color development therefore may vary with different proteins. Equipment: * Spectrophotometer, Σ960, (Metretech Inc, USA). * Automatic pipettes (Human, Japan) * Electric balance (Ohaus,USA) * Vortex mixer (Scientific Industries, China). Reagents and buffers: 1) Working solution: 40
  57. 57. Materials and Methods The following 2 stock solutions were prepared: Solution (1): 2 % Sodium carbonate (Na2CO3) (ADWIC) in 0.1 N Sodium hydroxide (ADWIC). Solution (2): 0.5 % Copper sulfate (CuSO45H2O) (ADWIC) in 1% Sodium potassium tartarate (ADWIC) then, mix 98 ml of solution (1) with 2 ml of solution (2). 2) Standard protein solution: Serial concentrations of bovine serum albumin (BSA)(Sigma Chemical Co., St. Louis, Missouri, USA), from 0.5 to 4.000 mg /ml of phosphate buffered saline (PBS), pH 7.2, were used to establish the standard calibration curve. 3) Blank solution: 0.1 M PBS (pH 7.2): was used as a blank solution. It prepared by dissolving the following components in 1L distilled H2O (7.4 gm NaCl + 0.51 gm NaH2PO4. 2H2O + 1.8 gm Na2HPO4). Adjust the pH to the desired value by using concentrated HCl. 4) Folin Ciocalteu’s reagent (1N): 2 N Folin reagent (Sigma) was diluted (V/V) using distilled H2O before use. Procedure: The antigenic solutions (20 µl) were added separately per 100 µl of working solution (standard protein was tested in parallel). Samples were mixed 41
  58. 58. Materials and Methods well using a vortex mixer and allowed to stand at room temperature (RT) for 10 min. Aliquots of 10 µl of 1 N Folin and Ciocalteu’s reagent were added to each tube then the contents were mixed well using the vortex and allowed to stand at RT for 30 min. A blue color was developed and the absorbance value was read at 490 nm using ELISA reader. A standard calibration curve was plotted using serial concentration of the standard BSA protein. The unknown concentrations of the antigenic solutions were determined from the curve, shown in figure 3. Figure 3. Standard calibration curve of bovine serum albumin. 4. Sodium dodocyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE): BCG, ascites fluid, CSF and serum samples at 30 µg/lane were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 42
  59. 59. Materials and Methods according to (Laemmli, 1970). A) Electrophoresis of proteins: Equipment: A- Dual minigel unit (Hoefer Scientific, USA) containing: 1) Glass plates (8.2 cm × 10.2 cm). 2) Plastic spacers (0.75 mm thickness). 3) Plastic combs (10 wells, 0.75 mm thickness). 4) Inner cooling core. 5) Clamp assemblies. 6) Lower buffer chamber. 7) Electrodes. B- Power supply (Hoefer Scientific, USA): Reagent and Buffers: - Gel acrylamide stock solution (30%): 29.2 gm acrylamide + 0.8 gm N, N-methylene bis - acrylamide (Sigma). -Sodium dodecyl sulfate (SDS) stock solution: 10% SDS (Sigma). - Resolving buffer: 0.03 M Tris-HCl, pH 8.3 (Sigma). - Stacking buffer: 0.65 M Tris-HCl, pH 6.8 (Sigma). - Tetramethylene diamine (TEMED), (Sigma). - Ammonium persulfate (Sigma). - Resolution buffer, pH 8.3: 0.192 M glycine, 0.02M Tris and 0.1% SDS. - Sample buffer: 20% of 0.5 Tris - HCl, pH 8.6 + 20% of glycerol (50%) + 5% SDS (10%) + 5% of bromophenol blue (1 %). - Molecular weight markers (Sigma) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55.0 kDa), ovalbumin (42.7 kDa), aldolase (40.0 kDa), Carbonic anhydrase (31.0 kDa), 43
  60. 60. Materials and Methods Soybean trypsin inhibitor (21.5 kDa). Procedure: 1. Preparation of resolving gel (12%): A. Glass-plate sandwich was assembled using two clean glass plates and two 0.75 mm spacers. B. The resolving (separating) gel was prepared by mixing the following: * 1.675 ml Distilled water. * 1.25 ml Resolving buffer. * 2.00 ml Acrylamide monomer. * 50 µl 10% SDS. * 25 µl 10% ammonium persulfate. * 2.5 µl TEMED c- The gel was poured between the glass plates immediately the gel top was carefully covered with 1cm of distilled water, then the gel was kept at RT for about 15 min. to polymerize. 2. Preparation of stacking gel (4%): A- the staking gel by was prepared mixing the followings: * 1.224 ml distilled water. * 0.5 ml stacking buffer. * 0.26 ml acrylamide monomer . * 20 µl 10% SDS. * 10µl 10% ammonium persulfate. * 2 µl TEMED. B-The water layer was poured off above the polymerized resolving gel and rinsed with few milliliters of stacking gel solution. The comb was aligned in the proper position then the stacking gel solution was added up to 2 mm from the top edge of the resolving gel. 44
  61. 61. Materials and Methods C- Resolving gel solution was left for polymerization for about 15 min. at RT. 3- Sample preparation: BCG, ascites fluid, CSF and serum samples at 30 µg/lane were mixed with sample buffer. The mixture was then boiled in water bath for 2 min. The mixture was then applied to the gel wells. 4- Running condition: Electrophoresis was carried out with constant volt of 200 V. The run was terminated when the bromophenol blue marker reach to the bottom of the gel. B) Staining of proteins: The separated proteins on polyacrylamide gel stained with coomassie blue R-250. Coomassie Blue staining requires an acidic medium for the generation of an electrostatic attraction between the dye molecules and the amino groups of the proteins. This ionic attraction, together with van der Waals forces, binds the dye-protein complex together. The binding is fully reversible by dilution under appropriate conditions. Coomassie stains give a linear response up to 20 mg/cm. however, the relationship between stain density and protein concentration varies for each protein (Andrews, 1986). Reagents: - Coomassie blue R-250 (Sigma). - Absolute methanol (BDH). - Acetic acid (ADWIC). Staining solution: 0.2 gm Coomassie blue R-250 was dissolved in a mixture of 80 ml of 40% methanol and 20 ml of 10 % acetic acid. Destaining solution: 40 % methanol + 10 % acetic acid. Procedure: The electrophoresis gel was soaked in excess of staining solution for 30 min.with constant shaking., The gel was rinsed with distilled H2O and destained 45
  62. 62. Materials and Methods with excess amount of destaining solution for several times with constant shaking until the excess stain was satisfactorily removed. 5. Immunoblotting Technique (Western Blot): In order to determine the TB target antigen for the (TB-55 mAb) in BCG, ascites fluid, CSF and serum samples, the separated proteins by SDS - PAGE were transferred from the polyacrylamide gel to nitrocellulose (NC) sheet according to the method of Towbin et al., (1979). A) Electroblotting: Equipment and materials: -Blotting apparatus (Hoefer, Scientific, USA). -Nitrocellulose filter (0.45µm) (Sigma,USA). Procedure: The gel, nitrocellulose sheets, sponge sheets and Whitman filter papers were equilibrated for 15 min. in transfer buffer pH, 8.3. (1.44 gm glycine + 0.303 gm Trisma base in 20% absolute methanol). The blotting sandwich was assembled within the blotting cassette. The cassette was inserted into blotting (transfer) buffer and the power supply was connected, as the cathode should be on the gel side. The blotting was carried out with constant voltage of 60 V for 2 hours. B) Immunostaining using TB-55 mAb Reagents and buffers: 1- Tris buffered saline (TBS), pH 7.4: 12.11 gm trisma base + 11.688 gm Sodium chloride were dissolved in 46
  63. 63. Materials and Methods 800 ml distilled water and the pH was adjusted to 7.4 using HCl. Then, the volume was completed to one liter with distilled water. 2- Blocking buffer: 5% (W/V) dry non-fat milk: 5 gm non-fat milk were dissolved in 0.05 M tris buffer, containing 0.15 M NaCl (TBS), pH 7.4. 3- Antigen: BCG, ascites fluid, CSF and serum samples from TB infected patients and ascites fluid, CSF and serum samples from non-infected individuals were used. 4- Primary antibody: An IgG monoclonal antibody (TB-55 mAb) was diluted 1: 150 in blocking buffer. 5- Secondary antibody: Anti-mouse IgG alkaline phosphatase conjugate (Sigma) was prepared in TBS, pH 7.4 in a dilution 1: 350. 6- Alkaline phosphatase substrate (BCIP/NBT): Premixed 5-Bromo-9-Chloro-3-Indolyl Phosphate (BCIP) / Nitro blue tetra-zolium (NBT), system pH 9.5 (ABC Diagnostics, New Damietta City, Egypt.) Procedure: The nitrocellulose filter (NC) was blocked in blocking buffer. The NC filter was then rinsed in TBS and incubated with (TB-55 mAb) with constant shaking overnight then washed in TBS three times, 10 min. each. The NC filter was incubated with goat anti-mouse IgG alkaline phosphatase conjugate, for 2 hours with dilution of (1: 350) followed by washing in TBS as mentioned before. The target antigen for TB-55 mAb was visualized by incubating the NC filter in substrate solution (BCIP/NBT) system. Then the reaction was stopped 47
  64. 64. Materials and Methods by distilled water. C) Molecular weight determination: The migration distances traveled by each protein starting from the top of resolving gel when divided by the distance traveled by the tracking dye gave relative mobility of the protein which known as (Rf). The standard molecular weight were plotted in terms of their Rf values, then Rf of the unknown protein was calculated and its molecular weight was determined from the blotted linear calibration figure. Rf = Migrated distance of protein Migrated distance of dye 6. Purification of the 55 kDa antigen from serum samples, ascites fluid and CSF of tuberculosis patients. Preparative gel electrophoresis: The analytical SDS-PAGE can be adapted for preparative purposes by increasing the thickness of gel. Preparative gels would ideally be capable of yielding high individual proteins recovered from corresponding analytical gels (Garfin, 1990). 1. Equipments & Reagents: The same as described under SDS-PAGE section except the use of plastic spacers (1.5 mm) to increase gel thickness. Procedure: The same as described in analytical SDS-PAGE. 48
  65. 65. Materials and Methods B. Electroelution: Electrophoretic separation of proteins in various types of polyacrylamide gels is employed from the analytical to the preparative scale. After separation, it is frequently necessary to extract or elute specific protein from the gel for further study. Electroelution is more controlled than diffusive elution and can be performed either during or after electrophoresis (Dunbar, 1990). Equipment: - Power supply (Sigma, USA) - Electroeluter unit (HYBAID) - Dialysis sacks (Sigma) MW CO : 12 – 14 kDa Flat width : 35 mm Inflat diameter : 31 mm Length : 30 mm Buffers and reagents: - Electroelution Buffer: 0.191 M glycine, 0.024 M Tris and 0.003 M SDS (Sigma). - Trichloroacetic acid 40 % (sigma) - Diethyl ether (BDH) - 0.01 M (PBS), pH 7.2 Procedure: A strip at one side of the electrophoresis preparative gel was cut and stained with Commie Brilliant Blue R - 250 as described before. After staining the strip is placed beside the unstained preparative gel and a band containing the wanted antigen was cut. The unstained strip containing the desired antigen was 49
  66. 66. Materials and Methods placed in a dialysis membrane with sufficient electroelution buffer volume and the antigen was eluted from the gel by electroelution with a constant volt of 200 v for 3 hrs. C. Dialysis against phosphate buffered saline: Electroeluted antigens from ascites fluid, CSF and serum of pulmonary and extra-pulmonary tuberculosis patients were dialysed against one liter of phosphate buffered saline (PBS), pH 7.2 overnight at 4 °C with constant stirring. D. Pre-concentration of the purified Antigen: After the dialysis step, the electroeluted antigens were concentrated using 50 ml of polyethylene glycol for 1 hour at room temperature. For further concentration the antigens were precipitated using 40% TCA final concentration (V/V) centrifuged at 6500 Xg for 15 min. The precipitates were washed twice using diethyl ether to remove the execs of TCA. The excess diethyl ether was removed by drying and the pellet was reconstituted in PBS, pH 7.2. and stored at-20° C until used. 7. Capillary Electrophoresis (CE): Capillary electrophoresis (CE) is a fully automated and computercontrolled powerful separation technique for biomolecules such as proteins. The method described by Attallah et al., (2003) was used for the separation of purified antigen with some modifications. 1. Equipments: Prince autosampler model 1-LIFT (Prince Technologies, Handelsweg, Emmen, The Netherlands), a programmable injector for capillary electrophoresis was used for the analysis. The instrument equipped with a high voltage supply that delivered up to 200 µA at voltage range 0 to 30 kV. The instrument 50
  67. 67. Materials and Methods connected with Lambda 1010 variable UV (Deuterium lamp)/VIS (Halogen lamp) detector (Metrhom Herisau, Switzerland). The instrument controlled by an IBM compatible computer fitted with WinPrince software, version 5 (Prince Technologies) running under Microsoft Windows 3.11 (Microsoft, WA, USA). Fused silica capillary (65 cm x 75 µm, i.d.,) coated with polyimide film (Prince Technologies) was used. Analyzed sample introduced into the capillary using Electrokinetic injection by applying high voltage and small pressure for few seconds. After injection, electrophoretic separation was performed using high voltage and the temperature controlled at 20 ± 0.1 oC. Detection was performed and signals analyzed using the Dax software, version 5 (Prince Technologies). 2. Reagents: Rinse solution (0. 1M Sodium hydroxide): 0.4 gm Sodium hydroxide dissolved in 100-ml distilled water. 0.1 M Hydrochloric acid: 8.660 ml of conc. HCl (11.6 N) diluted with 91.340 ml distilled water. Elution buffer (0.05 M Borate buffer, pH 8.3): 0.4 gm boric acid and 0.3 gm Sodium borate decahydrate (Borax, Na2B4O7. 10 H2O) were dissolved in 90-ml distilled water, the pH was adjusted using 0.1M HCl and volume completed to 100-ml using distilled water. 3. Running conditions: The purified antigens (25 µg) diluted with 0.5-ml distilled water and subjected to CZE. Before the analysis, the capillary was rinsed with 0.1 M NaOH for 30 seconds. Then, the capillary conditioned with Borate buffer (pH 8.3) for 60 seconds. The sample (10 µl) injected through the capillary by high 51
  68. 68. Materials and Methods voltage (30 kV) and low pressure (25 mbar) for 10 seconds. Then, sample eluted with Borate buffer (pH 8.3) by applying constant 30 kV voltage for 15 min. During separation, internal capillary temperature was constant at 20 oC during separation. Detection was performed by UV absorption at 200 nm. The signals were analyzed using Dax software, version 5 (Prince Technologies). 8. Biochemical characterization of the 55 kDa antigen from serum samples of pulmonary, extra-pulmonary, ascites fluid and CSF of tuberculosis patients : The methods described by Attallah et al., (2003) were used for identify nature of the antigens (protein, glycoprotein polysaccharide, etc) the antigen from ascites fluid, CSF and serum of pulmonary and extra-pulmonary tuberculosis patients were subjected to different biochemical treatments. A. NaOH treatment: Reagents: 0.2 N NaOH Procedure: One mg/ml of the purified antigens from ascites fluid, CSF and serum of pulmonary and extra-pulmonary tuberculosis patients were incubated with the same volume of 0.2 N NaOH for one hour at room temperature. After incubation tested using dot- ELISA after neutralized the mixture by 0.2 N HC1. B. HC1 treatment: Reagents: 0.2 M HCl Procedure: One mg/ml of purified antigens from ascites fluid, CSF and serum of pulmonary and extra-pulmonary tuberculosis patients were incubated with the 52