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Diagnosis of Mycobacterium Tuberculosis
 

Diagnosis of Mycobacterium Tuberculosis

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Mycobacterium Tuberculosis , diagnosis

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    Diagnosis of Mycobacterium Tuberculosis Diagnosis of Mycobacterium Tuberculosis Document Transcript

    • 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
    • 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
    • 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
    • ‫ﺑﺴﻢ اﷲ اﻟﺮﲪﻦ‬ ‫اﻟﺮﺣﯿﻢ‬ ‫"رب اﺷﺮح ﱃ ﺻﺪرى‬ ‫وﯾﺴﺮﱃ أﻣﺮى‬ ‫واﺣﻠﻞ ﻋﻘﺪة ﻣﻦ‬ ‫ﻟﺴﺎﱏ ﯾﻔﻘﻬﻮا‬ ‫ﻗﻮﱃ"‬ ‫ﺻﺪق اﷲ اﻟﻌﻈﯿﻢ‬
    • 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
    • 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.
    • 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.
    • 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
    • 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
    • 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 2.3.2.1. Types of extra pulmonary tuberculosis 13 2.3.2.1.1. Lymph node tuberculosis 13 2.3.2.1.2. Tuberculosis peritonitis 13 2.3.2.1.3. Genitourinary tuberculosis 14 2.3.2.1.4. Orthopedic tuberculosis 15 2.3.2.1.5. Miliary tuberculosis 16 2.3.2.1.6. Tuberculosis of the central nervous system 17 2.3.2.1.7. Tuberculosis of sinusitis 19 2.3.2.1.8. Other sites 19 2.4. Control of tuberculosis 19 21 3. Diagnosis of tuberculosis 3.1. Microscopic method 21 iii
    • Title Page 3.1.1. Ziehl - Neelsen staining technique 21 3.1.2. Auramine phenol fluorochrome staining technique 24 3.2.1.3. 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • Review of literature Figure 1. Epidemiology of TB in the world (WHO, 2000). 9
    • 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
    • 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
    • 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
    • Review of literature 2.3.2.1. Types of extra pulmonary TB 2.3.2.1.1. 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). 2.3.2.1.2. 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
    • 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). 2.3.2.1.3. 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
    • 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). 2.3.2.1.4. 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
    • 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). 2.3.2.1.5. 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
    • 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). 2.3.2.1.6. 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
    • 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
    • Review of literature 2.3.2.1.7. 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). 2.3.2.1.8. 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
    • 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
    • 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
    • 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
    • Review of literature Figure 2. Acid fast bacilli (shown in red) are tubercle bacilli (Grange, 2002) 23
    • 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
    • Review of literature 3.2.1.3. 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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). 3.6.1.1. 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). 3.6.1.2. 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). 3.6.1.3. 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • Materials and Methods same volume of 0.2 M HCl for one hour at room temperature. After incubation tested using dot ELISA after neutralized the mixture by 0.2 N NaOH. C. Sodium periodate treatment: Reagent: Sodium-m- periodate (20 mM) in PBS pH 7.2. Procedure: One mg/ml of the purified antigens from ascites fluid, CSF and serum of pulmonary and extra-pulmonary tuberculosis patients were oxidized with (20 mM) Sodium-m-periodate in PBS (pH 7.2) and the reaction mixture was kept in dark for 18 hour. Adding an equal volume of 130 mM glycerol then inhibited the reaction. The mixture was tested using dot ELISA. D. Mercaptoethanol treatment: Reagent: Mercaptoethanol (180 M) in PBS pH 7.2. Procedure: One mg/ml of the purified antigens from ascites fluid, CSF and serum of pulmonary and extra-pulmonary tuberculosis patients were treated with (180M) Mercaptoethanol in PBS (pH 7.2) and the reaction mixture was kept for one hour. The mixture was tested using dot ELISA. E. Trichloroacetic acid (TCA) treatment: Reagents: 40 % TCA 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 40 % TCA. After incubation at room temperature for 30 53
    • Materials and Methods minutes, the mixture was centrifuged at 10000 r.p.m for 15 minutes and then the precipitate was reconstituted with PBS. After that the supernatant and precipitate were tested using dot ELISA. F. Pepsin treatment: Reagents: Pepsin (Sigma). Procedure: One mg/ml of the purified antigens from ascites fluid, CSF and serum of pulmonary and extra-pulmonary tuberculosis patients were incubated with one mg/ml of pepsin for one hour at 37 °C. After incubation the mixture was tested by dot ELISA. G. Protease enzyme treatment: Reagents: Protease (Sigma). Procedure: One mg/ml of the purified antigens from ascites fluid, CSF and serum of pulmonary and extra-pulmonary tuberculosis patients were incubated with one mg/ml of protease for one hour at 37 °C. After incubation the mixture was tested by dot ELISA. 9. Amino acid analysis: Amino acid analysis provides an important quantitative parameter in the 54
    • Materials and Methods characterization of isolated proteins or peptide samples (Aitken and Learmonth, 1996). Equipment: High performance liquid chromatography system (HPLC)(Kontron co.) consisted of: -Two 322 solvent –delivery pumps automated by gradient system controller. -A model 742-HPLC detector system with variable wave length. -A spherisorb c8 column (250 x 4.6 mm id., kontron, Switzerland). Procedures: Sample Hydrolysis: One ml of purified antigen (1.0 mg/ml) was hydrolyzed under vacuum for 24 hr at 120 °C in 6 N HCl. Standard Preparation: A mixture of 17 amino acid standards were prepared in 0.1 N HCl and used for calibration. Chromatographic separation of amino acids: The hydrolysate (20 µl) was dried and derivatized by phenylisothiocyanate for 20 min. at room temperature. The derivatized amino acids were reconstituted with 200 µl of PBS (pH, 7.2). After vortex and sonicating for a few seconds, 10 µl was injected. The standards mixture of amino acid sample was treated as similar to the hydrolysate sample. The mobile phase consisted of a gradient of two eluents: Eluent (A) was an aqueous buffer of 0.1 M sodium acetate containing 1 PPM EDTA titrated to pH 5.5 with glacial acetic acid. Eluent (B) was an organic phase consisting of acetonitrile: methanol: water (45:40:15). The gradient employed in the separation started with eluent (B) rising from 6 to 45% in 60 min. A constant flow-rate of 1.5 ml/min was 55
    • Materials and Methods maintained through out. 10. Dot-ELISA: Dot-ELISA of Attallah et al., (2003) as a simple and rapid assay was used to detect the TB circulating 55-kDa antigen in serum samples of pulmonary and extrapulmonary tuberculosis using specific IgG monoclonal antibody. Reagents and buffers: I) Reagents: 1.Nitrocellulose membrane filter (0.45 µm, Sigma). 2. Bovine serum albumin (BSA); (Sigma). 3.IgG mouse monoclonal antibody (TB-55 mAb) 4.Anti-mouse IgG alkaline phosphatase, conjugate; (Sigma) 5. 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: All the following steps run out on the surface of nitrocellulose membrane filter fixed in plastic cartilage (Device). The nitrocellulose membrane surface weted by 0.1M PBS then 500 µg serum sample was added on the membrane. The nitrocellulose membrane surface was washed using 0.1M PBS three times then blocked using 5% BSA in 0.1M PBS. The nitrocellulose membrane surface was washed using 0.1M PBS three times. Monoclonal antibody diluted 1: 500 in 0.01 M PBS (pH 7.4) was applied. The nitrocellulose membrane surface was washed using 0.1M PBS three times. The second antibody, alkaline phosphatase conjugated goat antibody to mouse immunoglobulins diluted in 0.05 M Tris 56
    • Materials and Methods buffer. The nitrocellulose membrane surface was washed using 0.1M PBS three times. NBT/BCIP substrate working solution was added. After two minutes of adding substrate solution the reaction stopped by adding 100 µl of distilled H2O, then the result was taken. 11. Statistical Analysis: All statistical analyses were done by a statistical software package “SPSS 12.0 for windows, SPSS Inc.) Data were expressed as arithmetic mean ± standard deviation (X ± SD). The diagnostic sensitivity, specificity, efficiency, and positive predictive (PPV) and negative predictive (NPV) values were calculated as following: Reference Evaluated test Test Total + ve - ve + ve True + ve (a) False –ve (c) a+c - ve False + ve (b) True -ve (d) b+d Total a+b c+d a+b+c+d Where: Sensitivity: Sensitivity defined as the capacity of a certain technique of detecting the greatest number of individuals truly sick. Sensitivity = a / (a +c) × 100. Specificity: Specificity is the capacity of the test being always negative in the absence of the disease, not offering false-positive results. 57
    • Materials and Methods Specificity = d / (b + d) ×100. Efficiency = (a + d)/(a + b + c + d) ×100. Positive predictive value = a / (a +b) ×100. The positive predictive value indicates the probability that a patient with a positive test results has, in fact, the disease in question. Negative predictive value = d / (c +d) ×100. The negative predictive value indicates the probability that a patient with a negative test does not has the disease in question. 58
    • Results Results Part 1 Identification of the TB target antigen in Bacilli Calmette-Guerin (BCG) and different body fluids (serum,CSF and ascites): 1.1. SDS-PAGE and Western blot for BCG. BCG vaccine as an antigen of M. tuberculosis was analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and stained with coomassie blue (figure 4 A). The coomassie blue separated polypeptides have a wide range of molecular weights ranged from 120 kDa to 30 kDa. The separated proteins were electrophoretically transferred to nitrocellulose (NC) paper and immunostained with the specific mouse mAb designated TB–55 mAb. The TB-55 mAb identified two reactive bands in BCG vaccine, Figure 4B. 59
    • Results Figure 4. SDS-PAGE and Western blot analysis of BCG vaccine. A. SDS-PAGE: BCG vaccine at 30 µg/lane was resolved in 12 % SDS-PAGE and stained with coomassie blue. B. Immunoblot: The TB-55 mAb identified two reactive bands. Molecular weight markers (Mr.) 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) and Soybean trypsin inhibitor (21.5 kDa). 60
    • Results 1.1.1. Molecular weight determination of two reactive epitopes for the TB 55 mAb: To determine the molecular weight of two reactive epitopes for TB 55 mAb in BCG vaccine, linear calibration represents a relation between the molecular weight of protein standards mixture and their flow rates on SDS-PAGE was constructed, table 2 and figure 5. The flow rates of the reactive bands were calculated and their molecular weights were determined from the liner calibration. The molecular sizes of the reactive bands were 55-kDa and 82 kDa in BCG vaccine, figure 5. 61
    • Results Table 2. Rf values of unknown antigens and of standards protein mixture. Molecular weight (kDa) Log Molecular weight Rf values 97.4 2.99 0.12 66.2 1.82 0.24 55.0 1.74 0.31 42.7 1.63 0.45 40.0 1.6 0.50 31.0 1.49 0.71 21.5 1.33 0.96 1.91 0.14 1.74 0.31 Unknown antigen (82 kDa) Unknown antigen (55 kDa) 62
    • Results Phosphorylase B Bovine serum albumin Glutamate dehydrogenase Ovalbumin Aldolase Carbonic anhydrase Soybean trypsin inhibitor Figure 5. Liner calibration of standard molecular weights. 63
    • Results 1.2. Identification of TB-55 mAb target circulating antigen in different body fluids (serum,CSF and ascites): 1.2.1. SDS-PAGE and Western blot for sera from pulmonary tuberculosis patients and non infected individuals. Serum samples from patients infected with M. tuberculosis and non infected individuals, were analyzed by 12% one-dimensional (SDS-PAGE) under reducing conditions and staining with coomassie blue. The coomassie blue stained separated polypeptides have a wide range of molecular weights ranged from 97.4 kDa to 21.5 kDa as shown in figure 6. The separated proteins were electrophoretically transferred to nitrocellulose (NC) paper. TB–55 mAb was used as a primary antibody, and anti-mouse IgG alkaline phosphatase was used as a secondary antibody. The BCIP/NBT system was used as enzyme substrate. An intense sharp band in serum samples of pulmonary tuberculosis patients at 55-kDa but no reaction with non infected samples were observed as shown in figure 7. 64
    • Results Figure 6. Coomassie blue stained SDS-PAGE of sera from pulmonary tuberculosis patients and non infected individuals under reducing conditions. Serum samples at 30 µg/lane were loaded per well and electrophoresed under 200 volts for 45 minutes. Lanes (1-4): serum sample of non infected individuals. Lanes 5-9: serum samples from pulmonary tuberculosis patients. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa) and Soybean trypsin inhibitor (21.5 kDa). 65
    • Results Figure 7. Immunoblots of TB-55 mAb target antigen in sera of pulmonary tuberculosis patients and non infected individuals. Serum samples at 30 µg/lane were resolved in 12 % SDS-PAGE and electroblotted onto NC for 2 hours at 60 volts. The TB-55 mAb identified 55 kDa. Anti-mouse IgG alkaline phosphatase was used as a secondary antibody. BCIP/NBT system was used to visualize the reaction. Lanes (1-4): serum samples of non infected individuals. Lanes (5-9): serum samples from pulmonary tuberculosis patients. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa) and Soybean trypsin inhibitor (21.5 kDa). 66
    • Results 1.2.2. Identification of TB-55 mAb target circulating antigen in serum sample of extrapulmonary tuberculosis. 1.2.2.1. SDS-PAGE and Western blot for sera from extra-pulmonary tuberculosis patients and non infected individuals. Serum samples from extra-pulmonary tuberculosis patients (The sites of extra-pulmonary tuberculosis were peritonitis, meningitis, lymph nodes, genitourinary tract, Pott's disease, arthritis, sinusitis and milliary tuberculosis) and non infected individuals, were analyzed by 12% (SDS-PAGE) under reducing conditions and stained with coomassie blue. The coomassie blue stained separated polypeptides have a wide range of molecular weights ranged from 97.4 kDa to 21.5 kDa as shown in figure 8. The separated proteins were electrophoretically transferred to nitrocellulose (NC) paper. TB–55 mAb was used as a primary antibody, and anti-mouse IgG alkaline phosphatase was used as a secondary antibody. The BCIP/ NBT system was used as enzyme substrate. An intense sharp band corresponding to an antigen with 55 kDa was observed in serum samples of extra-pulmonary tuberculosis patients (The sites of extra-pulmonary tuberculosis were peritonitis, meningitis, lymph nodes, genitourinary tract, pott's disease, arthritis, sinusitis and milliary tuberculosis) but no reaction with non infected samples were observed as shown in figure 9. 67
    • Results Figure 8. Coomassie blue stained SDS-PAGE of sera from extra-pulmonary tuberculosis patients and non infected individuals under reducing conditions. Serum samples at 30 µg/lane were loaded per well and electrophoresed under 200 volts for 45 minutes. Lane (1): serum sample of non infected individual. Lane 2: peritonitis, Lane 3: meningitis, Lane 4: lymph nodes, Lane 5: genitourinary tract, Lane 6: Pott's disease, Lane 7: arthritis, Lane 8: sinusitis, Lane 9: milliary tuberculosis. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa), and Soybean trypsin inhibitor (21.5 kDa). 68
    • Results Figure 9. Immunoblots of TB-55 mAb target antigen in sera of extra pulmonary tuberculosis patients and non infected individuals. Serum samples at 30 µg/lane were resolved in 12 % SDS-PAGE and electroblotted onto NC for 2 hours at 60 volts. The TB-55 mAb identified a 55 kDa antigen. Lane (1): serum sample of non infected individuals. Lane 2: peritonitis, Lane 3: meningitis, Lane 4: lymph nodes, Lane 5: genitourinary tract, Lane 6: Pott's disease, Lane 7: arthritis, Lane 8: sinusitis, Lane 9: milliary tuberculosis. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa), Soybean trypsin inhibitor (21.5 kDa). 69
    • Results 1.2.3. Identification of TB-55 mAb target antigen in cerebrospinal fluid (CSF) of tuberculous meningitis. 1.2.3.1. SDS-PAGE and Western blot for CSF. CSF samples of tuberculous meningitis patients and non-tuberculous CSF were analyzed by 12% (SDS-PAGE) under reducing conditions and staining with coomassie blue. The coomassie blue stained separated polypeptides have a wide range of molecular weights ranged from 97.4 kDa to 55 kDa as shown in figure 10. The separated proteins of CSF samples were electrophoretically transferred to nitrocellulose (NC) paper. TB–55 mAb was used as a primary antibody, and anti-mouse IgG alkaline phosphatase was used as a secondary antibody. The BCIP/NBT system was used as enzyme substrate. An intense sharp band corresponding to an antigen with 55 kDa was observed in CSF from tuberculous meningitis patients but no reaction with non-tuberculous CSF from nontuberculous neurological diseases patients from control observed as shown in figure 11. 70 individuals were
    • Results Figure 10. Coomassie blue stained SDS-PAGE of CSF from tuberculous meningitis patients and non-tuberculous CSF under reducing conditions. CSF samples at 30 µg/lane were loaded per well and electrophoresed under 200 volts for 45 minutes. Lanes (1-3): non-tuberculous CSF from nontuberculous neurological diseases patients. Lanes (4-9): CSF from tuberculous meningitis patients. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa), Soybean trypsin inhibitor (21.5 kDa). 71
    • Results Figure 11. Immunoblots of CSF from tuberculous meningitis patients and non-tuberculous CSF using TB-55 mAb. CSF samples at 30 µg/lane were resolved in 12 % SDS-PAGE and electroblotted onto NC for 2 hours at 60 volts. The TB-55 mAb identified 55 kDa antigen. Lanes (1-3): non-tuberculous CSF. Lanes (4-9): CSF from tuberculous meningitis patients. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa), Soya bean trypsin inhibitor (21.5 kDa). 72
    • Results 1.2.4. Identification of TB-55 mAb target antigen in tuberculous ascetic fluid: 1.2.4.1. SDS-PAGE and Western blot for ascetic fluid. Tuberculous ascetic fluid samples from peritonitis tuberculosis patients and non-tuberculous ascites (transudative and exudative) were analyzed by 12% SDS-PAGE under reducing conditions and stained with coomassie The coomassie blue stained separated polypeptides have a wide range of molecular weights ranged from 97.4 kDa to 21.5 kDa as shown in figure 12. The separated proteins of ascetic fluid samples were electrophoretically transferred to nitrocellulose (NC) paper. TB–55 mAb was used as a primary antibody, and anti-mouse IgG alkaline phosphatase was used as a secondary antibody. The BCIP/ NBT system was used as enzyme substrate. An intense sharp band corresponding to an antigen with 55 kDa was observed in tuberculous ascetic fluid from peritonitis tuberculosis patients but no reaction with non-tuberculous ascites fluids (transudative and exudative) individuals were observed as shown in figure 13. 73 from control
    • Results Figure 12. Coomassie blue stained SDS-PAGE of tuberculous ascetic fluid from peritonitis tuberculosis patient and non-tuberculous ascites fluid under reducing conditions. Ascetic fluid samples at 30 µg/lane were loaded per well and electrophoresed under 200 volts for 45 minutes. Lane (1): transudative ascites from non-tuberculosis ascites patient, lane 2: exudative ascites from non-tuberculosis ascites patient. Lanes (3-9): tuberculous ascetic fluids from peritonitis tuberculosis patients. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa), Soybean trypsin inhibitor (21.5 kDa). 74
    • Results Figure 13. Immunoblots of TB-55 mAb target antigen in tuberculous ascetic fluid from peritonitis tuberculosis patients and non-tuberculous ascites fluids. Ascetic fluid samples at 30 µg/lane were resolved in 12 % SDSPAGE and electroblotted onto NC for 2 hours at 60 volts. The TB-55 mAb identified 55 kDa. Anti-mouse IgG alkaline phosphatase was used as a secondary antibody. BCIP/NBT system was used to visualize the reaction. Lane (1): transudative ascites from non-tuberculosis ascites patient, lane 2: exudative ascites from non-tuberculosis ascites patient. Lanes (3-9): tuberculous ascetic fluids from peritonitis tuberculosis patients.. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa), Soybean trypsin inhibitor (21.5 kDa). 75
    • Results Part 2 Purification and characterization of the circulating 55 kDa antigen from different body fluids of pulmonary and extrapulmonary tuberculosis patients. 2.1. Purification of the circulating 55 kDa antigen from different body fluids of pulmonary and extra-pulmonary tuberculosis patients. The 55-kDa antigen was purified from different body fluids of pulmonary and extra-pulmonary tuberculosis patients using electroelution technique from preparative slab gels. The purified 55-kDa antigen from different body fluids was treated with tricholoroacetic acid (TCA) and the precipitate fraction was analyzed by 16 % SDS-PAGE and stained with coomassie stain. The results showed that the precipitate of purified antigen from different body fluids of pulmonary and extra-pulmonary tuberculosis patients revealed a single polypeptide chain at 55-kDa as shown in figures (14-17). The purified antigen from serum of pulmonary and extra-pulmonary, ascites and CSF of extra-pulmonary tuberculosis patients showed a single peak when analyzed by capillary zone electrophoresis at 11 minutes as shown in figure 18 a, b, c, d. 76
    • Results Figure 14. Coomassie stain SDS-PAGE of purified 55 kDa antigen from sera of pulmonary patients under reducing conditions. Purified antigen was loaded at 30 µg/lane per well and electrophoresed under 200 volts for 45 minutes. Lane 1: crude serum sample from pulmonary tuberculosis patient. Lane 2: The precipitated fraction of the purified antigen from pulmonary tuberculosis patients treated with TCA. Lane 3: The supernatant fraction of the purified antigen from pulmonary tuberculosis patients treated with TCA. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa), Soybean trypsin inhibitor (21.5 kDa). 77
    • Results Figure 15. Coomassie stain SDS-PAGE of purified 55 kDa antigen from sera of extra-pulmonary tuberculosis patients under reducing conditions. Purified antigen was loaded at 30 µg/lane per well and electrophoresed under 200 volts for 45 minutes. Lane 1: crude serum sample from extra-pulmonary tuberculosis patient. Lane 2: The precipitated fraction of the purified antigen from extra-pulmonary tuberculosis patients treated with TCA. Lane 3: The supernatant fraction of the purified antigen from extra-pulmonary tuberculosis patients treated with TCA. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa), Soybean trypsin inhibitor (21.5 kDa). 78
    • Results Figure 16. Coomassie stain SDS-PAGE of purified 55 kDa antigen from CSF under reducing conditions. Purified antigen was loaded at 30 µg/lane per well and electrophoresed under 200 volts for 45 minutes. Lane 1: crude CSF fluid. Lane 2: The precipitated fraction of the purified antigen from CSF treated with TCA. Lane 3: The supernatant fraction of the purified antigen from CSF treated with TCA. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa), Soybean trypsin inhibitor (21.5 kDa). 79
    • Results Figure 17. Coomassie stained SDS-PAGE of purified 55 kDa antigen from ascites under reducing conditions. Purified antigen was loaded at 30 µg/lane per well and electrophoresed under 200 volts for 45 minutes. Lane 1: crude ascites fluid. Lane 2: The precipitated fraction of the purified antigen from ascites treated with TCA. Lane 3: The supernatant fraction of the purified antigen from ascites treated with TCA. Molecular weight markers (Mr.) include: Phosphorylase B (97.4 kDa), Bovine serum albumin (66.2 kDa), Glutamate dehydrogenase (55 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), Carbonic anhydrase (31 kDa), Soybean trypsin inhibitor (21.5 kDa). 80
    • Results A Figure 18 a. Capillary electrophoresis electropherogram of purified 55 kDa antigen from pulmonary tuberculosis. The purified antigen from serum of pulmonary patients showed a single peak when analyzed by capillary zone electrophoresis at 11 minutes. The purified 55 kDa antigen (25 µg per one ml of distilled water) separated with 100-mM borate buffer, pH 8.3, on a 65-cm x 75µm capillary, 30 kV, 20 oC and UV detection at 200 nm. 81
    • Results B. Purified antigen from extra-pulmonary tuberculosis B Figure 18 b. Capillary electrophoresis electropherogram of purified 55 kDa antigen from sera of extra-pulmonary tuberculosis. The purified antigen from serum of extra-pulmonary patients showed a single peak when analyzed by capillary zone electrophoresis at 11 minutes. The purified 55 kDa antigen (25 µg per one ml of distilled water) separated with 100-mM borate buffer, pH 8.3, on a 65-cm x 75-µm capillary, 30 kV, 20 oC and UV detection at 200 nm. 82
    • Results C Figure 18 c. Capillary electrophoresis electropherogram of purified 55 kDa antigen from CSF. The purified antigen from CSF showed a single peak when analyzed by capillary zone electrophoresis at 11 minutes. The purified 55 kDa antigen (25 µg per one ml of distilled water) separated with 100-mM borate buffer, pH 8.3, on a 65-cm x 75-µm capillary, 30 kV, 20 oC and UV detection at 200 nm. 83
    • Results D Figure 18 d. Capillary electrophoresis electropherogram of purified 55 kDa antigen from ascites fluid. The purified antigen from ascites fluid showed a single peak when analyzed by capillary zone electrophoresis at 11 minutes. The purified 55 kDa antigen (25 µg per one ml of distilled water) separated with 100-mM borate buffer, pH 8.3, on a 65-cm x 75-µm capillary, 30 kV, 20 oC and UV detection at 200 nm. 84
    • Results 2.2. Reactivity of the purified 55 kDa antigen against TB-55 monclonal antibody. The TB 55 mAb was used as a probe in dot-ELISA. Color dot corresponding to the purified antigen was observed in TCA reconstituted precipitated fractions of serum samples of pulmonary and extrapulmonary, ascites and CSF of patients with extra-pulmonary tuberculosis but no reaction with TCA supernatant fractions was observed in serum samples of pulmonary and extrapulmonary, ascites and CSF of patients with extra-pulmonary tuberculosis as shown in figure 19. 85
    • Results Figure 19. Reactivity of the purified 55 kDa antigen against TB-55 monclonal antibody using dot-ELISA. Positive (+ve) control : Serum sample of infected individuals with M. tuberculosis reactive with TB-55 mAb on Western blot technique. Negative (-ve) control : Serum sample of non-infected individuals with M. tuberculosis not reactive with TB-55 mAb on Western blot technique. A1-A4: The TCA precipitates of the purified fraction from serum samples of pulmonary (A1) and extra-pulmonary (A2) tuberculosis patients, CSF (A3) and ascetic fluid (A4). B1- B4: The TCA supernatant of the purified fraction from serum samples of pulmonary (B1) and extra-pulmonary (B2) tuberculosis patients, CSF (B3) and ascetic fluid (B4). 86
    • Results 2.3. Partial biochemical characterization of the purified 55-kDa antigen reactive epitope isolated from different sources (serum, CSF, ascites): The characterization of the reactive epitope of the purified 55-kDa antigen recognized by TB-55 mAb was carried out by exposing the purified antigen to various reagents such as acid, alkali, tricholoroacetic acid (TCA), periodate, mercaptoethanol, protease and pepsin enzymes. The epitope reactivity of the purified antigen against TB-55 mAb was tested using dot ELISA. The reactive epitope of the purified antigen from different sources has the same biochemical characters. The results showed that the reactivity of the antigen was lost after treatment with acid, alkali, mercaptoethanol, protease and pepsin enzymes but was maintained after periodate treatment. Antigen precipitated with TCA showed reactivity against TB55-mAb in contrast to the supernatant that did not show reactivity. Also the purified antigen fractions were treated with constant concentration of protease and pepsin enzymes. The enzymatic reaction was stopped at different time intervals (15, 30, 45 min). The reactivity of the purified antigen was tested against TB55-mAb using dot ELISA. The results showed that the reactivity of the purified antigen was decreased with increasing the incubation time of protease and pepsin enzymes but the reactivity was completely lost after 45 min as shown in table 3. 87
    • Results Table 3. Partial biochemical characterization of the reactive epitope on purified 55-kDa antigen isolated from different sources (serum, CSF, ascites) . Reactivity of Treatments purified antigen using dot-ELISA Incubation Type of reagents Treated Untreated Concentrations time Acid 0.2 M HCl 1 hour -Ve + Ve Base 0.2 M NaOH 1 hour -Ve + Ve 40% 15 min. a- precipitate + Ve - b- Supernatant -Ve - Trichloroacetic acid Periodate oxidation 20 mM 18 hours + Ve + Ve Mercaptoethanol 180 M 1 hour -Ve + Ve Protease enzyme 1 mg/ml 45 min. -Ve + Ve Pepsin enzyme 1 mg/ml 45 min. -Ve + Ve -Ve: Negative reaction +Ve: Positive reaction 88
    • Results 2. 4. Amino acid analysis of the purified 55-kDa antigen: The purified antigen was hydrolyzed with 6 N HCl at 110 °C overnight. The amino acid compositions of the 55-kDa antigen was analyzed using high performance liquid chromatography (HPLC). The results showed that the 55kDa of M. tuberculosis antigen consisted of 15 amino acids (leucine, isoleucine, valine, proline, methionine, tyrosine, alanine, glycine, serine, theronine, lysine, arginine, histidine glutamic and aspartic). The hydrophobic amino acids (leucine, isoleucine, valine, proline methionine, tyrosine and alanine) represented 24.6% while the hydrophilic amino acids (glycine, serine and theronine) represented 46.4%. Basic amino acids (lysine, arginine and histidine) represented 16.3% and acidic amino acids (glutamic and aspartic) represented 12.7% as shown in table 4 and figure 20. So, the 55- kDa antigen is a basic polypeptide chain with a hydrophilic nature. 89
    • Results Table 4. Amino acid concentrations of the purified 55 kDa M. tuberculosis antigen. Concentration % Type Name Leucine Proline 59.28 98 Tyrosine 94.28 Alanine 113.14 Glycine 755.71 Serine 214.28 Therionine 55.71 Lysine 162.85 Arginine 85.71 Histidine Acidic 81.42 Methionine Basic 49.28 Valine Hydrophilic 47.14 Isoleucine Hydrophobic (nmol/mg protein) 111.42 Glutamic acid 135.71 Aspartic acid 142.85 90 24.6 % 46.4 % 16.3 % 12.7 %
    • Percentage (%) Results 50 45 40 35 30 25 20 15 10 5 0 46.4 % 24.6 % Hydrophobic 16.3 % 12.7 % Hydrophilic Basic Acidic Types of amino acides Figure 20. The relative percentages of the amino acid concentrations of the purified 55 kDa antigen. Hydrophobic amino acids are leucine, isoleucine, valine, proline, methionine, tyrosine and alanine. Hydrophilic amino acids are glycine, serine and theronine. Basic amino acids are lysine, arginine and histidine. Acidic amino acids are glutamic and aspartic. So, the 55- kDa antigen is a basic polypeptide chain with a hydrophilic nature. 91
    • Results Part 3 Evaluation of simple and rapid detection of circulating 55 kDa antigen using dot ELISA. 3.1. Types of tuberculosis included in the present study. Serum samples of 506 individuals were included in the present study. They included patients with pulmonary TB (n= 296), patients with extra-pulmonary TB (n= 93) as well as sera of patients with respiratory diseases other than TB (n= 69 ) and healthy controls (n= 48). Of the 389 cases examined, 296 were pulmonary tuberculosis (76 %) and 93 cases (24 %) were extra-pulmonary tuberculosis, figure 21. Patients with extra pulmonary TB (n= 93) consist of 25 TB peritonitis (27 %), 22 TB meningitis (24 %), 19 genitourinary tract (20 %), 14 TB lymphadenitis (16 %), 5 Pott's disease (5 %), 3 TB arthritis (3 %), 3 TB sinusitis (3 %), 2 milliary TB (2 %) as listed in table 5. 92
    • Percentage (%) Results 100 90 80 70 60 50 40 30 20 10 0 76 % 24 % Pulmonary TB Extra-Pulmonary TB Types of tuberculosis Figure 21. Types of tuberculosis. Of the 389 tuberculosis cases examined, 296 were pulmonary tuberculosis (76 %) and 93 cases (24 %) were extrapulmonary tuberculosis. 93
    • Results Table 5. The types of extra-pulmonary tuberculosis according to sites of infection in 93 serum samples. Types of extra-pulmonary tuberculosis No. % TB peritonitis 25 27 TB meningitis 22 24 TB of genitourinary tract 19 20 TB lymphadenitis 14 16 Pott's disease 5 5 TB sinusitis 3 3 TB arthritis 3 3 Milliary TB 2 2 94
    • Results 3.2. Detection of circulating 55- kDa in serum samples using dot-ELISA: TB-55 mAb antibody was used as a probe in dot-ELISA to detect a target tuberculosis antigen in serum according to Attallah et al., (2003). This is a semi-quantitative assay, requires no sophisticated equipment, rapid (5 minutes) and requires little or no skill to perform without pretreatment of serum sample and the results can be read visually without the need of ELISA reader. An intense sharp violet color was observed in serum samples of tuberculosis infected patients but no reaction with non-infected individuals serum samples was observed. The developed violet color varied in its intensity, from weak (1+ or, 2+) to strong (3+ or, 4+). Colorless dot (negative test) was produced in case of no antigen detection, i.e., negative test as shown in figure 22. 95
    • Results Figure 22. Dot-ELISA of serum samples from tuberculosis patients and non-infected individuals. The assay showed different antigen levels according to the developed color. Positive (+ve) control : Serum sample of infected patient with M. tuberculosis reactive with TB-55 mAb on Western blot. Negative (-ve) control : Serum sample of non-infected individual with M. tuberculosis not reactive with TB-55 mAb on Western blot. Strong positive test : Serum samples with high antigen level (3+, 4+). Weak positive test : Serum samples with low antigen level (1+,2+). 96
    • Results 3.3. Detection of circulating 55- kDa in serum samples of pulmonary tuberculosis patients using dot-ELISA. Serum samples of patients with pulmonary TB (n= 257) were tested for circulating 55- kDa using dot-ELISA. Of 296 pulmonary tuberculosis cases, 257 were positive for TB antigen (87 %) and 39 cases (13 %) were negative for TB antigen. Levels of circulating 55-kDa antigen using Dot- ELISA in serum samples of pulmonary patients. 39 out of 296 pulmonary tuberculosis (13 %) were negative, 215 (73%) were positive with low antigen level and 42 (14 %) were positive with high antigen level, figure 23. To evaluate the efficiency of the Dot-ELISA for the detection of TB circulating 55-kDa antigen in serum samples of pulmonary tuberculosis patients. The antigen was detected in 257 out of 296 serum samples of pulmonary tuberculosis patients with sensitivity (87 %). 113 sample out of 117 patients with respiratory diseases other than TB and healthy individuals (controls) were negative for the antigen with 97 % specificity, 90 % efficiency, positive predictive value (98 %), negative predictive value (74 %), table 6. 97
    • Results 80 73% 70 Percentage % 60 50 40 30 14% 13% 20 10 0 Negative Low Antigen level* High Antigen level** Figure 23. Levels of circulating 55-kDa antigen detection using Dot- ELISA in serum samples of pulmonary tuberculosis patients. * Weak positive test (+/++) ** Strong positive test (+++/++++) 98
    • Results Table 6. Advantages of circulating 55-kDa antigen detection by using DotELISA in serum samples of pulmonary tuberculosis. TB-Ag detection Clinical diagnosis (gold standard) Total + 257 (a) 39 (c) 296 4 (b ) 113 (d ) 117 261 Pulmonary tuberculosis patients - 152 413 Patients with respiratory diseases other than TB and Healthy controls Total Sensitivity = a / (a +c) × 100= 257/296 ×100 = 87 %, Specificity = d / (b + d) ×100 = 113/117 ×100 = 97 %, Efficiency = (a + d)/(a + b + c + d) ×100 = 370 /413 × 100 = 90 %, Positive predictive value = a / (a +b) ×100 = 257/261 × 100 = 98 %, Negative predictive value = d / (c +d) ×100 = 113/152 = 74 % 99
    • Results 3.4. Detection of circulating 55- kDa in serum samples of extra-pulmonary tuberculosis patients using dot-ELISA. Serum samples of patients with extra pulmonary TB (n= 93) were tested for circulating 55- kDa using dot-ELISA. Of 93 cases, 84 were positive for TB antigen (90 %) and 9 cases (10 %) were negative for TB antigen. Detailed analysis of extra-pulmonary tuberculosis using dot ELISA were listed in table 7. 22 out of 25 TB peritonitis were positive for circulating 55kDa (88 %), 20 out of 22 TB meningitis were positive for circulating 55- kDa (91%), 17 out of 19 of TB genitourinary tract were positive for circulating 55kDa (89%), 12 out of 14 of TB lymphadenitis were positive for circulating 55kDa (86 %), 5 out of 5 Pott's disease were positive for circulating 55- kDa (100 %), 3 out of 3 of TB sinusitis were positive for circulating 55- kDa (100 %), 3 out of 3 of TB arthritis were positive for circulating 55- kDa (100 %), 2 out of 2 of milliary TB were positive for circulating 55- kDa (100 %). Levels of circulating 55-kDa antigen using Dot- ELISA in serum samples of extra-pulmonary patients were calculated 9 out of 93 extra-pulmonary tuberculosis (10 %) were negative, 58 (62 %) were positive with low antigen level and 26 (28 %) were positive with high antigen level , figure 24. 100
    • Results To evaluate the efficiency of the Dot-ELISA for the detection of TB circulating 55-kDa antigen in serum samples. The antigen was detected in 84 out of 93 serum samples of extra-pulmonary tuberculosis patients with sensitivity (90 %). 113 sample out of 117 patients with respiratory diseases other than TB and healthy individuals (controls) are negative antigen with 97 % specificity, 94 % efficiency, positive predictive value (95 %), negative predictive value (93 %) as shown in table 8. 101
    • Results Table 7. Detailed analysis of extra-pulmonary tuberculosis using dot ELISA. Types of extra-pulmonary No. of No. +ve % +ve tuberculosis serum samples samples TB peritonitis 25 22 88 TB meningitis 22 20 91 TB genitourinary tract 19 17 89 TB lymphadenitis 14 12 86 pot's disease 5 5 100 TB sinusitis 3 3 100 TB arthritis 3 3 100 Milliary TB 2 2 100 102
    • Results 70 62 % 60 Percentage % 50 40 28% 30 20 10% 10 0 Negative Low Antigen level* High Antigen level** Figure 24. Levels of circulating 55-kDa antigen detection using Dot- ELISA in serum samples of extra-pulmonary tuberculosis patients. * Weak positive test (+/++) ** Strong positive test (+++/++++) 103
    • Results Table 8. Advantages of circulating 55-kDa antigen detection by using DotELISA in serum samples of extra-pulmonary tuberculosis. TB-Ag detection Clinical diagnosis (gold standard) Total + 84 (a) 9 (c) 93 4 (b ) 113 (d ) 117 88 Extra-pulmonary tuberculosis patients - 122 210 Patients with respiratory diseases other than TB and Healthy controls Total Sensitivity = a / (a +c) × 100= (84/93) ×100 = 90 %, Specificity = d / (b + d) ×100 = 113/117 ×100 = 97 %, Efficiency = (a + d)/(a + b + c + d) ×100 = 197/210 × 100 = 94 %, Positive predictive value = a / (a +b) ×100 = 84/88 × 100 = 95 %, Negative predictive value = d / (c +d) ×100 = 113/122 = 93 % 104
    • Results 3.6. Overall levels and advantage of TB-circulating 55-kDa antigen detection by using dot- ELISA in serum samples. Overall levels of circulating 55-kDa antigen using Dot- ELISA in serum samples of pulmonary and extra-pulmonary patients. 48 out of 389 pulmonary and extra-pulmonary tuberculosis (12.3 %) were negative, 273 (70.2 %) were positive with low antigen level and 68 (17.5 %) were positive with high antigen level, figure 25. To evaluate the efficiency of the Dot-ELISA for the detection of TB circulating 55-kDa antigen in serum samples. The antigen was detected in 341 out of 389 serum samples of tuberculosis patients with sensitivity (88 %). 113 sample out of 117 patients with respiratory diseases other than TB and healthy individuals (controls) are negative antigen with 97 % specificity, 90 % efficiency, positive predictive value (99 %), negative predictive value (70 %) as shown in table 9 and figure 26. All samples showing false negative results (n= 48) using dot-ELISA were tested using the more sensitive Western blot and 55-kDa antigen was detected in all (100 %) false negative samples. 105
    • Results 80 70.2 % 70 Percentage % 60 50 40 30 20 17.5 % 12.3 % 10 0 Negative Low Antigen level* High Antigen level** Figure 25. Overall levels of circulating 55-kDa antigen detection using DotELISA in serum samples of pulmonary and extra-pulmonary tuberculosis patients. ** Weak positive test (+/++) *** Strong positive test (+++/++++) 106
    • Results Table 9. Overall Advantages of circulating 55-kDa antigen detection by using Dot- ELISA in serum samples. Clinical diagnosis (gold standard) TB-Ag detection Total + 341 (a) 48 (c) 389 4 (b ) 113 (d ) 117 345 TB patients - 161 506 Patients with respiratory diseases other than TB and Healthy controls Total Sensitivity = a / (a +c) × 100= 341/389 ×100 = 88 %, Specificity = d / (b + d) ×100 = 113/117×100 = 97 %, Efficiency = (a + d)/(a + b + c + d) ×100 = 454 /506 × 100 = 90 %, Positive predictive value = a / (a +b) ×100 = 341/345 × 100 = 99 %, Negative predictive value = d / (c +d) ×100 = 113/161 = 70% 107
    • Results 99 % 97 % 100 88 % 90 % 90 70 % Percentage % 80 70 60 50 40 30 20 10 0 Sensitivity Specificity Efficiency PPV* NPV** Figure 26. Overall Advantages of circulating 55-kDa antigen detection by using Dot- ELISA in 506 serum samples. PPV* =Positive predictive value NPV** = Negative predictive value 108
    • Discussion V. Discussion Despite the discovery of the tubercle bacillus more than a hundred years ago, and all the advances in our knowledge of the disease made since then, tuberculosis still remains one of the major health problems facing mankind, particularly in developing countries (Gradmann, 2006). Presently, about one third of the world, s population is infected with M.tuberculosis. It is estimated that currently there are about 10 million new cases of tuberculosis every year with 3 million deaths occurring world-wide. Currently, more people die of tuberculosis than from any other infectious disease. Death from tuberculosis comprises 25% of all avoidable deaths in developing countries. Nearly 95% of all tuberculosis cases and 98% of deaths due to tuberculosis are in developing countries and 75% of tuberculosis cases are in the economically productive age (Ramachandran and Paramasivan, 2003). M. tuberculosis causes pulmonary tuberculosis, and the clinical manifestations of infection can be either acute, or latent and asymptomatic, depending on the intensity of the immune response mounted by the infected patient. After being exposed to M. tuberculosis, 40% of the individuals that become infected will develop primary active tuberculosis, and 60% remain with the latent form of the bacilli and may present extrapulmonary sites of infection, resulting from inefficient macrophage action at the beginning of exposure (Beck et al., 2005). The standard diagnosis is still made by clinical examination, direct sputum microscopy, and bacterial culture (Nahid et al., 2006). However, tuberculosis does not always present the classic radiological signs that allow an easy diagnosis, especially in extra-pulmonary cases. The traditional laboratory methods used for complementation of diagnosis have their limits, such as low sensitivity of acid fast smears, the time needed for cultivation, with undetectable growth in only 10 to 20% of the cases, and the high costs involved in molecular detection methods, such as polymerase chain reaction (Beck et al., 109
    • Discussion 2005). The detection of Mycobacterial DNA in clinical samples by polymerase chain reaction is a promising approach for the rapid diagnosis of tuberculous infection (Nahid et al., 2006). However, the PCR results must be corrected for the presence of inhibitors as well as for DNA contamination (Garg et al., 2003). Many studies have focused on the detection of antibodies specific for different M. tuberculosis antigens that indicate active disease. Such a rapid serologic test should be economic and successful in cases where the classical methods are not sufficient. M. tuberculosis-circulating antigen in clinical specimens from pulmonary tuberculosis patients have been made by several authors (Stavri et al., 2003 and Attallah et al., 2003). In the present study, Western blot analysis revealed that TB-55 mAb reacted against an antigen at an apparent molecular weight of 55 kDa in serum samples of pulmonary and extra-pulmonary tuberculosis patients, BCG vaccine, CSF and ascites fluid of infected patients and but no reaction was observed in serum samples, ascites fluid and CSF of controls. In addition to the 55kDa reactive epitope, a higher molecular weight epitope was identified at 82-kDa in BCG vaccine suggesting that the 55-kDa serum antigen may be the stable degradation product from the higher molecular weight antigen. However, further molecular study is required for confirmation. The specificity of TB-55mAb is borne out by the fact that it does not bind to antigens present in the body fluids of nontuberculous patients. It is of interest that an antigen with a similar size has not been previously reported in serum samples of extra-pulmonary tuberculosis. It is possible that such antigens could be shed directly into the infected area or may arise from sequestered Mycobacterium in tissues. Regardless of the mechanism by which these antigens appear in body fluids, the present study indicates its effective diagnostic potential. 110
    • Discussion Many of investigators have used BCG as an antigen source and relied upon commercial antisera for the detection of M. tuberculosis antigens in body fluids (Wadee et al., 1990). Theodora et al., (1991) purified and characterized ten major antigens from M. bovis culture filtrate of 39, 32, 30, 25, 24, 22, 19, 15, and 12 kDa by classical physicochemical methods. Freer et al., (1998) raised monoclonal antibodies against M.bovis bacillus Calmette-Guerin (BCG) culture filtrate proteins or live BCG. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Similarly, other investigators detected M. tuberculosis antigens in serum , ascites fluid CSF and different body fluids (Wadee et al., 1990).samples of tuberculosis patients. Ng et al., (1995) detected 30 kDa antigen in serum samples of fifty-one African patients with clinically diagnosed tuberculous pericardial effusion (of whom 25 had confirmation by pericardial fluid culture) using a monoclonal antibody and western immunoblotting. Attallah et al., (2003) identified a target mycobacterial circulating antigen of 55-kDa molecular weight in sera from confirmed M. tuberculosis infected individuals by using Western blotting based on a specific mouse IgG anti-M. tuberculosis monoclonal antibody TB-55 mAb. No bands were identified in sera of healthy individuals. Similarly, Wadee et al., (1990) detected 43 kDa circulating antigen in cerebrospinal fluid, pleural and ascitic fluid specimens using analyes of these body fluids by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and western immunoblotting and ELISA. Such antigens were not detected in body fluids of nontuberculous patients. 111
    • Discussion Similarly, other investigators detected antigen 5, and 14 kDa M. tuberculosis antigens in cerebrospinal fluid of tuberculous meningitis (Radhakrishnan and Mathai, 1991 and Sumi et al., 1999). Katti (2001) developed a reverse passive hemagglutination has using rabbit antimycobacterial IgG for detection of circulating mycobacterial antigens in CSF from chronic infections of the central nervous system. Immunoblot analysis of reverse passive hemagglutination positive CSF revealed predominantly 30-32 kDa and 71 kDa antigens whilst 6, 86, 120, 96 and 110 kDa showed varied degree of reactivity. Mathai et al., (2001) subjected heat-inactivated CSF specimens from tuberculous and non-tuberculous patients to sodium dodecyl sulfate polyacrylamide gel electrophoresis and they were subsequently transferred onto nitrocellulose membrane using a rabbit polyvalent antibody to M. tuberculosis, a heat stable 82 kDa mycobacterial antigen was demonstrated in the CSF of patients with tuberculous meningitis. This antigen was conspicuous by its absence in the CSF of non-tuberculous subjects. Kashyap et al., (2005) demonstrated the presence of a 30-kDa protein band in CSF of 100% (n=5) of confirmed and 90% (n = 138) of suspected tuberculous meningitis patients out of 153 tuberculous meningitis patients. Immunohistochemical staining procedure is a simple and sensitive technique which has been used to identify Mycobacterium in cultures, sputum as well as other smears and tissue sections. There are many immunoreactive substances within the cell wall and cytoplasm of Mycobacteria comprising proteins, polysaccharides and lipids. And these have been characterized and standardized at an international workshop (WHO, 1986). Earlier studies of immunohistochemical staining have shown the utility of polyclonal and monoclonal antibodies to identify M. tuberculosis antigens in the lung, brain and 112
    • Discussion lymph node and joint specimens of tuberculous patients too have helped arrive at an accurate diagnosis of tuberculosis (Ashok et al., 2002). Humphrey and Weiner, (1987) detected mycobacterial antigens in lung tissue specimens using an indirect peroxidase-antiperoxidase method and was compared to the detection of AFB by Ziehl-Neelsen stain. Histologic specimens were obtained from 59 hospital patients. Of nine patients with mycobacterial disease, seven had antigen detected in tissue. In two patients with tuberculous pneumonia, the distribution of mycobacterial antigens was approximately the same as that of AFB. In contrast, in four patients with caseating pulmonary granulomas, clumps of mycobacterial antigens were demonstrated in necrotic areas of the granulomas where there were few or no AFB. In one patient with M. intracellulare infection, cross-reactive antigens stained weakly. Antigen was not found in tissue from two patients; one had miliary lung granulomas, and the second had mediastinal lymph node granulomas. Mycobacterial antigens were not detected in specimens from 50 control patients with nonmycobacterial diseases. Barbolini et al., (1989) directed four monoclonal antibodies 60.15, 61.3, 105.10, and 2.16, to different proteins of M. tuberculosis using an indirect peroxidase method to detect mycobacterial antigens in lung, lymph node, and joint tissue specimens of tuberculous patients. Using monoclonal antibody 60.15, which recognizes protein with a molecular mass of 28 kDa. With MoAb 61.3, which reacts with a 35 kDa protein present in M. tuberculosis, M. africanum, and M. bovis. Monoclonal antibodies 105.10 and 2.16 bind to the cross-reactive 65 kDa heat shock protein that is present in mycobacteria and stain scattered particles and dark clumps of bacilli within the phagocyte cytoplasm. Sumi et al., (1999) standardized immunocytochemical method for the direct demonstration of mycobacterial antigen in cerebrospinal fluid specimens 113
    • Discussion of patients with tuberculous meningitis. CSF-cytospin smears were prepared from 22 patients with a clinical diagnosis of tuberculous meningitis and also from an equal number of patients with nontuberculous neurological diseases (disease control). Immunocytological demonstration of mycobacterial antigens in the cytoplasm of monocytoid cells was attempted, by using rabbit immunoglobulin G to M. tuberculosis as the primary antibody. Of the 22 CSFcytospin smears from tuberculous meningitis patients, 16 showed positive immunostaining, while all of the CSF-cytospin smears from the disease control showed negative immunostaining for mycobacterial antigen. Attallah et al., (2005) carried immunohistochemical staining using TB55 mAb for localization of target antigen in lymph tissues. Immunohistochemical staining showing different patterns of mycobacterial antigen distribution. Their distribution were seen as solid, beaded or fragmented rods, within phagocyte cytoplasm in areas without caseous necrosis. The second, diffuse staining in the form of antigenic dust was also seen in giant cells and epithelioid cell cytoplasm. Specimen of tuberculous lymphadenitis with omission of TB –55mAb were used as negative controls. In the present study, the lower molecular weight 55–kDa target antigen was purified from serum of pulmonary and extrapulmonary tuberculosis, ascites and CSF of patients with extra-pulmonary tuberculosis using electroelution from polyacrylamide preparative slab gels. The results showed that the precipitate of purified antigen from different body fluids of pulmonary and extra-pulmonary tuberculosis patients revealed a single polypeptide chain at 55-kDa and showed a single peak when analyzed by capillary zone electrophoresis at 11 minutes. The TB 55 mAb was used as a probe in dot-ELISA. A color dot corresponding to the purified antigen with 55 kDa was observed in TCA reconstituted precipitated fraction but no reaction with TCA soluble fraction was observed. The reactive epitope of the purified antigen was destroyed (i.e. showing negative 114
    • Discussion result using dot-ELISA) to acid and base hydrolysis, mercaptoethanol, protease, and pepsin treatments. The purified antigen were precipitated with 40% TCA, and reconstituted in PBS, pH 7.2. The reconstituted precipitated of purified antigen showed high reactivity (i.e. colored dot) toward TB-55 mAb. In contrast, the supernatant of purified antigen showed no reactivity (colorless dot). Periodate treatment did not affect the reactivity of the target epitope of purified antigen. So these purified antigen have the same biochemical nature as the purified antigen from sera of pulmonary tuberculosis patients (Attallah et al., 2003). Several investigators isolated circulating antigen M. tuberculosis antigens from serum samples of tuberculosis patients (Nair, 2000 and Banerjee et al., 2003). Nair, (2000) isolated circulating antigen from bacteriologically confirmed tuberculous sera by ammonium sulphate precipitation. The protein fraction between 36%, and 75%, ammonium sulphate was reactive with tuberculosis sera showing the presence of circulating tubercular antigen. Circulating tubercular antigen was seroreactive similar to 31 kDa antigen isolated from in vitro culture medium. Banerjee et al., (2003) isolated circulating antigen from confirmed pulmonary tuberculosis serum and bone and joint tuberculosis serum by trichloroacetic acid precipitation and further fractionation by fast-protein liquid chromatography. This antigen was seroreactive similarly to in vitro released 41 kDa antigen isolated from culture medium. Kashyap et al., (2005) excised 30-kDa band from the gel, destained extensively, and digested with trypsin. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry. Partially purified proteins from CSF samples of tuberculous meningitis were analyzed by two-dimensional polyacrylamide gel electrophoresis and Western blotting. Immunoblotting and 115
    • Discussion enzyme-linked immunosorbent assay (ELISA) were performed to confirm the presence of proteins in the 30-kDa protein band. Several investigators isolated M. tuberculosis antigens from culture of M. tuberculosis and characterized M. tuberculosis antigens, which may be protein such as 11.6 kDa, 30 kDa, 33 kDa, 38 kDa and 65 kDa or glycoprotein such as 45 kDa and or lipoproteins as 41 kDa (Zengyi et al., 1996; Salata et al., 1991; Deshpande et al., 1996; Kadival et al., 1987; Cummings et al., 1996 ; and Karen et al., 1995). Kadival et al., (1987) isolated 38 kDa antigen of M. tuberculosis by affinity chromatography using a monoclonal antibody. This antibody bound only to an antigen found in M. tuberculosis and M. bovis BCG. The antigen was detected only by antisera to M. tuberculosis and M. bovis. Salata et al., (1991) purified 30 kDa antigen of M. tuberculosis by ammonium sulfate precipitation, ion-exchange chromatography, and reversephase high-performance liquid chromatography to yield a single 29 to 30 kDa component. Immunoelectrophoresis studies demonstrated the purified 30 kDa antigen to be immunologically identical with antigen 6 and antigen 85B. The 30 kDa native antigen was a potent skin test antigen in sensitized guinea pigs. BY Lee et al., (1992) isolated and purified 19 kDa from enriched membrane fractions of the virulent Erdman strain of M. tuberculosis. Electron spray ionization mass spectrometry demonstrated a measured mass of 16,100, deviating from the predicted mass by only 2.86 atomic mass units. Immunoblotting indicated that this protein is highly expressed in the virulent strains of M. tuberculosis. Deshpande et al., (1994) purified 66-kDa protein from culture filtrate and cell sonicate of M. tuberculosis H37Rv by immobilised metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid column. TB66 was found 116
    • Discussion to be a fibronectin-binding protein as determined by ELISA and could be purified by affinity chromatography with fibronectin-Sepharose. A similar 66kDa protein could be isolated also from M. bovis, M. bovis BCG, M. africanum and M. tuberculosis H37Ra by IMAC, but not from any other Mycobacteria. Deshpande et al., (1996) isolated 33-kDa protein (TB33) from a delipidated cell sonicate of Mycobacterium tuberculosis H37Rv using immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid column. TB33 could not be isolated from the culture filtrate of M. tuberculosis H37Rv using nickel-nitrilotriacetic acid column. TB33 was recognized by monoclonal antibodies known to react with proteins of M. tuberculosis with a molecular mass of 33/34 kDa. Weldingh and Andersen (1999) purified and investigated six novel proteins in the region of 17-29 kDa for their immunological relevance in M. tuberculosis-infected mice, guinea pigs and tuberculosis patients. The proteins CFP17, CFP21, CFP25 and CFP29 were all identified as strong interferongamma inducers in M. tuberculosis-infected mice and in tuberculosis patients. Bhaskar et al., (2000) purified immunogenic antigen, CFP 6 was from culture filtrate of M. tuberculosis by a preparatory 2-D electrophoresis method. The protein focused at pI of 4.0 during isoelectric focusing. Molecular weight of the purified protein was 12 kDa. The occurrence of glycosylated proteins in M. tuberculosis has been widely reported. However, unequivocal proof for the presence of true glycosylated amino acids within these proteins has not been demonstrated, and such evidence is essential because of the predominance of soluble lipoglycans and glycolipids in all mycobacterial extracts (Karen et al., 1995). Espitia and Mancilla, (1989) identified three concanavalin A (ConA)binding bands of 55, 50 and 38 kDa in M. tuberculosis culture filtrates, by 117
    • Discussion labelling blotted proteins with a ConA-peroxidase conjugate. Binding was inhibited by the competitor sugar alpha-methyl mannoside and by reduction with sodium m-periodate. Bands of 55, 50 and 38 kDa stained with Coomasie blue were sensitive to digestion with proteases, thus indicating that they are proteins. Glycoproteins were isolated by lectin affinity chromatography or by elution from nitrocellulose membranes. On the isolated form, the 55-50 kDa doublet glycoprotein was 65.4% protein and 34.6% sugar. The purified 38 kDa molecule was 74.3% protein and 25.7% carbohydrate. By immunoblot, antibodies against mycobacterial glycoproteins were demonstrated in immunized rabbits and in patients with pulmonary tuberculosis, but not in healthy individuals. Treatment with sodium m-periodate abolished binding of rabbit antibodies to the 38 kDa glycoprotein. Reactivity of the 55-50 kDa doublet glycoprotein was not altered by reduction. By immunoblot with monoclonal antibodies TB71 and TB72, a carbohydrate-dependent and a carbohydrate-independent epitope could be identified on the 38 kDa glycoprotein. Avdienko et al., (1996) produced seven monoclonal antibodies against M. tuberculosis H37Rv. The mAb acted against M. tuberculosis H36Rv with molecular mass 14, 17-15, 25 27 30 kDa excluding monoclonal antibodies S5B3B8 and S3H5D7 which acted against the main antigen with 54 kDa mass and 5-6 bands of antigens. Chemical nature of antigenic determinants recognizable by a panel of monoclonal antibody was investigated. Mild sodium periodate oxidation and protease digestion of mycobacterial antigens showed that monoclonal antibody recognize both carbohydrate-containing epitopes and protein epitopes or protein and carbohydrate-containing antigenic determinants. In the present study, the amino acid analysis of the purified 55- kDa antigen, using high performance liquid chromatograph showed that the 55- kDa of M. tuberculosis antigen consisted of 15 amino acids (leucine, isoleucine, valine, proline, methionine, tyrosine, alanine, glycine, serine, theronine, lysine, 118
    • Discussion arginine, histidine glutamic and aspartic). The hydrophobic amino acids (leucine, isoleucine, valine, proline, methionine, tyrosine and alanine) represented 24.6% while the hydrophilic amino acids (glycine, serine and theronine) represented 46.4%. Basic amino acids (lysine, arginine and histidine) represented 16.3% and acidic amino acids (glutamic and aspartic) represented 12.7%. So, the 55- kDa antigen was basic polypeptide chain with a hydrophilic nature. Daniel and Anderson, (1978) purified M. tuberculosis antigen 5 from unheated culture filtrates by absorption onto an immunoabsorbent prepared with globulin from a monospecific goat antiserum and elution with 4.0 M urea at pH 9.0. The product was a homogeneous protein giving a single stainable band in gel electrophoresis and a single precipitin arc in immunoelectrophoresis. It was found to have a molecular weight of 28.5-35 kDa and a sedimentation constant of 2.0. Amino acid analysis demonstrated it to be rich in aspartic acid, suggesting a cytoplasmic origin. Yano et al., (1984) purified tuberculin-active substance, designated TAS1D3, has been from the extract of M. bovis BCG by precipitation at pH 4.2, ethanol fractionation, and column chromatography. TAS-1D3 was homogeneous in polyacrylamide gel electrophoresis and positive in both Coomassie brilliant blue and periodic acid-Shiff staining, suggesting that TAS-1D3 was a glycoprotein. The molecular weight of TAS-1D3 was estimated to be 26,000 by gel filtration. In amino acid analysis, TAS-1D3 was distinctive in having proline as a dominant amino acid, and in that it lacked basic amino acids, sulfurcontaining amino acids and aromatic amino acids. Karen et al., (1995) confirmed the presence of several putative glycoproteins in subcellular fractions of M. tuberculosis by reaction with the lectin concanavalin A. One such product, with a molecular mass of 45 kDa, was purified from the culture filtrate. Compositional analysis demonstrated that the 119
    • Discussion protein was rich in proline and that mannose, galactose, glucose, and arabinose together represented about 4% of the total mass. The standard methods used for diagnosis of tuberculosis had been to demonstrate microbiologically the presence of Mycobacterium tuberculosis in secretions and/or tissue from the patient. Improvements have been made that permit greater sensitivity for the examination of stained smears and for more rapid detection of growth of the organism using radiometric techniques. New methods for diagnosis that may well eliminate the need for smear and culture of specimens are under varying stages of development. These new methods are based on the detection of specific components of the organisms or on detection of specific antibodies produced by the patient. Some of these methods will require expensive and sophisticated equipment, and this will make them much less available in developing countries. The use of gene probes for diagnosis of TB is in use now on a limited scale (Crawford et al.,1989 and Ramachandran and Paramasivan , 2003). Monoclonal antibodies, provide the means to obtain a sensitivity and specificity to rival the tuberculin skin test and equal other commonly used diagnostic blood tests (Bothamley, 1995). Serum samples of 506 individuals were included in the present study. They included patients with pulmonary TB (n= 296), patients with extra-pulmonary TB (n= 93) as well as sera of patients with respiratory diseases other than TB (n= 69 ) and healthy controls (n= 48). Serum samples of the 389 tuberculosis patients were screened by the dot-ELISA. Of the 389 cases examined, 296 were pulmonary tuberculosis (76%) and 93 cases (24 %) were extra-pulmonary tuberculosis. 93 patients with extra pulmonary TB were classified according to the location of the infection as follow: peritonitis tuberculosis (27%), meningitis tuberculosis (24%), genitourinary tract (20 %), lymph nodes (16 %), potts disease (5%), arthritis (3 %), sinusitis (3%), millary (2%). 120
    • Discussion Several outhers reported different detection rate of pulmonary tuberculosis ranged from (60-89%) of tuberculosis cases, and different detection rate of each types of extra-pulmonary tuberculosis ranged from (11-40 %) (Gupta et al., 1995). Hayati et al., (1993) analyzed 100 cases of extrapulmonary tuberculosis were identified at the general hospital Kota Bharu representing 11% of all the newly diagnosed tuberculosis between January 1990 and December 1991. The sites involved were the lymph nodes (34%), osteoarticular (14%), miliary (12%) and pleura (10%). Fernandez et al. (1995) studied 107 cases of extrapulmonary tuberculosis diagnosed during a period lasting from 1988 to 1992 in a general hospital. These cases represent 35.7% from the overall tuberculosis diagnosed in the same period of time and same attendance centre. The most common forms of disease were tuberculosis pleural effusions (29%), genito-urinary (22%) and lymph node disease (20.5%). Rabaud et al., (1997) analyzed 351 files in nine voluntarily participating hospitals in France between January 1990 and December 1994. 79% of all cases were exclusively pulmonary, 14% were exclusively extra-pulmonary. Lado et al., 2000) observed a total of 921 tuberculosis infected patients, of which 370 (40.2%) were extrapulmonary forms. The distribution of extrapulmonary tuberculosis was: 307 extrapulmonary forms (83%) of which 140 (45.6%) were pleural, 87 (28.3%) ganglionary, 16 (5.2%) intestinal, 14 (4.5%) bone and joint, 11 (3.6%) genitourinary, 11 (3.6%) cutaneous, 10 (3.3%) meningeal, and other locations 18 (5.9); mixed forms 38 cases (10.3%); disseminated forms 8 cases (2.1%) and miliary TB 1 case (4.6%). In HIV infected patients 17 extrapulmonary forms (77.3%), which were mainly 121
    • Discussion ganglionary (64.7%); 4 disseminated forms (18.2 %); and 1 miliary TB (4.5%) cases were observed. Cagatay et al., (2004) analysed the incidence, clinical sites and risk factors for extrapulmonary tuberculosis in 252 patients with extrapulmonary tuberculosis between 1 January 1991 and 30 June 2003. Tuberculous lymphadenitis (36.5%) was found to be the most common clinical presentation of extrapulmonary tuberculosis. Nissapatorn et al., (2004) found that during a 2-year retrospective study, 195 patients with extrapulmonary tuberculosis were diagnosed at the National Tuberculosis Center, Kuala Lumpur, representing 10% of all patients with tuberculosis. The three main sites of involvement were lymph nodes (42.6%), miliary and disseminated (19.5%), and pleura (12.8%). Sensitive and specific techniques to detect and identify M. tuberculosis directly in clinical specimens are important for the diagnosis and management of patients with tuberculosis (Broccolo et al., 2003). Thus, new early and rapid diagnostic procedures are important for TB control (Martin, 2001). Simple diagnostic assays that are rapid, inexpensive, and do not require highly trained personnel or a complex technological infrastructure are essential for global control of tuberculosis (Samanich, 2000). Any test is to replace direct microscope must offer advantage in terms of speed and ease of use and preferably have a higher sensitivity. Antigen detection assays are promising in this regard, since they enable the analyst to test many samples at once (Lenka et al., 2000). In the present study, of 296 pulmonary tuberculosis cases, 257 were positive for TB antigen (87 %) and 39 cases (13 %) were negative for TB antigen. Levels of circulating 55-kDa antigen using Dot- ELISA in serum samples of pulmonary patients were negative (13 %), positive with low antigen level (73%) and positive with high antigen level (14 %). The antigen was 122
    • Discussion detected in serum samples of pulmonary tuberculosis patients with 87 % sensitivity, 97 % specificity, 90 % efficiency, positive predictive value (98 %), negative predictive value (74 %). The diagnostic potential of Mycobacterium antigen detection has been evaluated in serum (Sada, 1992) and sputum samples of pulmonary tuberculosis with sensitivity rates of 80-88% and specificity rate of 93-100%. However, none of these tests to detect mycobacterial antigens has become available for clinical utility nor achieved widespread use for the diagnosis of TB. Sada, (1992) established a coagglutination technique for the detection of lipoarabinomannan of Mycobacterium tuberculosis in human serum samples for its utility in the diagnosis. The coagglutination technique had a sensitivity of 88% in patients with sputum-smear-positive active pulmonary tuberculosis. The sensitivity in patients with active pulmonary tuberculosis negative for acid-fast bacilli in sputum was 67%. Less favorable results were obtained for patients with AIDS and tuberculosis, with a sensitivity of 57%. The specificity in control patients with lung diseases different from tuberculosis and in healthy subjects was 100%. The positive predictive value was 100%, and the negative predictive value for patients with sputum-positive active pulmonary tuberculosis was 97%. Several authors detected M. tuberculosis antigen in sputum samples for its utility in the diagnosis of pulmonary tuberculosis using ELISA (Yanez, 1986; Banchuin et al., 1990; Cho, et al., 1990; Al-Orainey et al., , 1992 and Pereira et al., 2000). Yanez (1986) developed double-antibody sandwich ELISA for detection mycobacterial antigens in sputum using a commercially available hyperimmune serum directed against BCG. A total of 68 unknown sputum specimens submitted to the clinical laboratories for examination for tuberculosis were tested by ELISA. Of the 20 specimens that were smear positive and culture 123
    • Discussion positive, 12 (60%) were positive by ELISA; 6 of the 11 (55%) smear-positive culture-negative samples were positive by ELISA; 1 of 2 (50%) of the smearnegative culture-positive samples was positive by ELISA; and only 3 of 35 (9%) of the smear-negative culture-negative samples were positive by ELISA. Banchuin et al., (1990) used a double antibody sandwich ELISA with commercially available anti-BCG and peroxidase labeled anti-BCG, for the detection of mycobacterial antigens in sputum samples. Positive results of ELISA were obtained from 24/25 sputum specimens which were positive for staining of acid fast bacilli, 5/16 specimens positive for culture of Mycobacterium tuberculosis and 67/69 specimens positive for both tests. The assay was positive in only 11/164 specimens negative for both staining of AFB and culture of M. tuberculosis. 4 of which were known to have tuberculosis. Thus, with sputum specimens, the sensitivity, specificity, efficiency, positive predictive value and negative predictive value of the ELISA were 87 %, 93 %, 90 %, 89 % and 91%; respectively. Cho, et al., (1990) developed ELISA for detecting mycobacterial antigen in sputum samples of pulmonary tuberculosis using the monoclonal antibodies. When 14 clinical specimens proven to contain AFB by smear staining or culture were examined, ten (71.4%) were positive by the sandwich ELISA; in contrast, sputum smear examination gave positive results in only six (42.9%) specimens. Meanwhile, none of 25 specimens with no evidence of AFB were positive by the sandwich ELISA. Al-Orainey et al., (1992) detected mycobacterial antigens in sputum using enzyme immunoassay. The system utilises commercially available antiBCG immunoglobulin. BCG protein standard was used as positive control. Thirty-nine patients with culture-proven pulmonary tuberculosis were tested. 124
    • Discussion The EIA was positive in 24 of 29 patients with positive smears and cultures, giving a sensitivity of 86 %. It was also positive in six of ten patients with smear-negative culture-positive disease, resulting in a sensitivity of 60% in this group. In another 176 patients with different nontuberculous pulmonary infections, only nine were positive by enzyme immunoassay, giving a specificity of 95 %. Pereira et al., (2000) developed capture enzyme-linked immunosorbent assay for detection of lipoarabinomannan in human sputum samples. As a capture antibody. A murine monoclonal antibody against Lipo arabinomannan (LAM), with rabbit antiserum against Mycobacterium tuberculosis as a source of detector antibodies. Thirty-one (91%) of 34 sputum samples from 18 Vietnamese patients with tuberculosis (32 smear positive and 2 smear negative) were positive in the LAM detection assay. In contrast, none of the 25 sputum samples from 21 nontuberculous patients was positive. Several authors detected M. tuberculosis antigen in sputum samples for diagnosis of pulmonary tuberculosis using dot ELISA (Kansal and Khuller, 1991; Deodhar et al., 1998 and Stavri et al., 2003) with different sensitivity and specificity. Kansal and Khuller, (1991) develop a simple and economical dot ELISA for the detection of mannophosphoinositide antigen in sputum samples of tuberculosis patients has been developed using affinity-purified antibodies. This test is able to detect free as well as bound antigen. Sputum samples from 94 patients suffering from tuberculosis and 30 non-tuberculosis patients were screened and an overall sensitivity and specificity of 89% and 93.3%, respectively, was obtained. Deodhar et al., (1998) developed a simple dot (blot) ELISA test for detecting tubercular antigen in sputum samples of patients of pulmonary 125
    • Discussion tuberculosis has been standardized using nitrocellulose paper. Of the 1042 patients in the study group, the percentage positivity by smear and culture was 54 % and 57% respectively; 68% of the ELISA positives were confirmed by smear. The dot blot ELISA could be used as a rapid and specific test as it not only picked up 89% of the smear positive, culture positive cases but also 82 % of the smear negative, culture positive cases. Stavri et al., (2003) detectd mycobacterial antigens in clinical specimens from pulmonary tuberculosis patients using enzyme immunoassay. 87 sputa, 87 sera and 40 paired sputa and sera were utilized from smear-positive and smear-negative, culture-positive patients; 59 sputa, 37 sera and 22 paired sputa and sera from nontuberculosis respiratory disease patients and 68 sera from healthy controls. The antigen detection in sputum by dot-ELISA has 86 % sensitivity on active tuberculosis patients, 92 % specificity, 91 % positive predictive value, 88 % negative predictive value and 10 % error. Extra-pulmonary TB is often difficult to diagnose because of its diverse clinical presentations (Walsh and McNerney, 2004). The most affordable diagnostic methods for the clinical setting are the immunoassays, since it is rapid, easy to perform and require simple reagents. Many serological assays have been developed for specific antibody detection in TB patients (Khomenko et al., 1996; Stavri et al., 2003). However, people in the tropical areas are in contact with various pathogens and developed cross-reacting antibodies responsible for poor specificity (Rasolofo and Chanteau 1999). Moreover, the sensitivity of antibody detection tests is much lower in HIV seropositive patients co-infected with tuberculosis (Ratanasuwan et al., 1997). Recently, more efforts are directed toward developing reliable, and less costly immunoassays based on the detection of mycobacterial antigens in different body fluids using specific antibodies. Such tests could be useful for the diagnosis and follow-up of TB patients (Pereira et al., 2000). Several M. 126
    • Discussion tuberculosis antigens were detected in different body fluids of infected individuals, e.g., 30-kDa antigen and 31-kDa antigen in serum (Ng et al.,1995; Nair et al., 2001), 43-kDa antigen in ascetic fluid (Wadee et al., 1990), 43-kDa antigen, antigen 5, and 14 kDa antigen in CSF (Radhakrishnan and Mathai , 1991 and Aggarwal et al., 2001). The diagnostic potential of Mycobacterium antigen detection has been evaluated in serum (Ng et al.,1995; Ashok et al., 2002 and Lenka et al., 2000), ascetic fluid (Wadee, 1990) and CSF (Srivastava et al. 1998 and Mathai et al., 2003) samples of extrapulmonary tuberculosis patients, with sensitivity rates of 41-93% and specificity rate of 86100%. However, none of these tests to detect mycobacterial antigens has become available for clinical utility nor achieved widespread use for the diagnosis of TB (Attallah et al., 2005). In the present study, serum samples of patients with extra pulmonary TB (n= 93) were tested for circulating 55- kDa using dot-ELISA. Of 93 cases, 84 were positive for TB antigen (90 %) and 9 cases (10 %) were negative for TB antigen. The detection rate of extra-pulmonary tuberculosis using dot ELISA were 88 %, 91%, 89%, 86 %, 100 %, 100 %, 100 % and 100 % in TB peritonitis, TB meningitis, TB genitourinary tract, TB lymphadenitis, Pott's disease, TB sinusitis, TB arthritis and milliary TB ; respectively. Levels of circulating 55-kDa antigen using Dot- ELISA in serum samples of extrapulmonary patients were negative (10 %), positive with low antigen level (62 %) and positive with high antigen level (28 %). The sensitivity, specificity, efficiency, positive predictive value, negative predictive value of the Dot-ELISA for the detection of TB circulating 55-kDa antigen in serum samples were 90 %,97 %, 94 % 95 % and 93 %; respectively. Furthermore, this study shows that the test can be used for the initial diagnosis of extra-pulmonary tuberculosis such as tuberculous peritonitis, 127
    • Discussion meningitis, lymphadentitis, genitourinary tract, potts disease, arthritis, millary tuberculosis and antigen can be detected even with a low antigenic load using non-invasive serum samples. The results clearly indicate that the test falsely detect only a few healthy controls individuals and patients with other diseases, thus giving it a high specificity. The Dot ELISA technique, because of its low cost, seems a viable alternative to the more expensive and sophisticated techniques (Rajpal et al., 2003). Radhakrishnan and Mathai, (1991) standardized a simple dotimmunobinding assay for diagnosis of tuberculous meningitis to detect M. tuberculosis antigen 5 and antimycobacterial antibody in cerebrospinal fluid specimens of patients with tuberculous meningitis. Sensitivity and specificity of Dot-Iba was compared with conventional ELISA and standard bacteriological techniques. The Dot-Iba showed excellent correlation with indirect ELISA for the detection of antimycobacterial antibody in CSF and showed 60% sensitivity and 100% specificity in culture-negative patients with tuberculous meningitis. However Dot-Iba was less sensitive for the detection of antigen 5 in CSF and showed false negative results (60%) in culture-positive patients with tuberculous meningitis. Although the ELISA system is very practical and sensitive, the testing equipment required is not always available in areas where tuberculosis is endemic. An alternative to ELISA could be the dot blot method, which uses only a paper matrix onto which the antigen is spotted, and the development of the antigen antibody reaction is done by an enzyme or the use of a colloidal gold conjugate (Stott, 1989). In addition, changes in antigen conformation that may occur as a result of passive coating of the antigens to solid supports may cause technical artifacts resulting in false-positive and false negative reactions (Pereira et al., 2003). 128
    • Discussion In the present study, overall levels of circulating 55-kDa antigen using Dot- ELISA in serum samples of pulmonary and extra-pulmonary patients. 48 out of 389 pulmonary and extra-pulmonary tuberculosis (12.3 %) were negative, 273 (70.2 %) were positive with low antigen level and 68 (17.5 %) were positive with high antigen level. The overall advantages of TB-circulating 55kDa antigen detection by using dot- ELISA in serum samples were evaluated. The antigen was detected in serum samples of tuberculosis patients with sensitivity (88 %), 97 % specificity, 90 % efficiency, positive predictive value (99 %), negative predictive value (70). All samples showing false negative results (n= 48) using dot-ELISA were tested using the more sensitive Western blot and 55-kDa antigen was detected in all (100 %) false negative samples. According to the recommendations of the World Health Organization, to replace the “gold standard”, bacterial culture, a serological test must possess a sensitivity of over 80% and specificity of over 95% (WHO, 1997). So detection of TB-circulating 55-kDa antigen using dot- ELISA in serum samples may replase M. tuberculosis culture. In the present study, the false negative results of the developed dot ELISA may be explained as follows. The 55 kDa circulating antigen level among false negative samples may be too low to be detected (Martin et al., 2001). In addition, The 55 kDa circulating antigen have been found as components of circulating immune complexes to achieve a higher sensitivity in the immunoassay (Doskeiand and Berdai, 1980 and De Jonge et al., 1987). In antigen detection assays, sample processing is often too laborious for daily use in laboratories in endemic areas and involves time consuming steps. However, the serum samples will be pretreated inactivates the antibodies and simultaneously, the antigens are released, and the epitopes are exposed. The false positive results of the developed dot ELISA may be explained as follows. The clinical diagnosis of tuberculosis is often problematic. A number of respiratory diseases such as 129
    • Discussion pneumonia, bronchitis, and cancer can mimic both clinical symptoms and the shadow often seen on a radiograph with pulmonary TB patients. Most patients with respiratory diseases other than showed negative results using dot- ELISA, although 3 % were positive. As all patients with respiratory disease other than TB were sputum negative , only radiographs and clinical symptoms were used for their diagnosis. Therefore, it is possible that concurrent TB infection could also be present in some patients with respiratory diseases other TB, diagnosed with circulating antigen detection, i.e. showing false positives (Jackkett et al., 1988; Attallah et al., 2003). In conclusion, we have identified the 55–kDa antigen in ascites fluid, CSF and serum using western blot technique. The 55–kDa antigen was purified from these fluids and showed a single band in comassie blue stained SDS-PAGE and one peak when analyzed by capillary zone electrophoresis at 11 minutes. The amino acid analysis of the purified 55- kDa antigen, using high performance liquid chromatograph, showed that the 55- kDa was basic polypeptide chain with a hydrophilic nature. The dot-ELISA detected the TB antigen in 90% sera of individuals with extra-pulmonary TB and in 87% sera of individuals with pulmonary TB. The overall sensitivity, specificity, efficiency, positive predictive value, negative predictive value of circulating 55-kDa antigen were 88 %, 97 % ,90 % 99 % and 70 % ; respectively. We have demonstrated that the Dot-ELISA method for tuberculosis antigen detection in pulmonary and extra-pulmonary tuberculosis could find practical application for the early laboratory diagnosis of tuberculosis, even in the laboratories with limited resources and technical expertise. Hence we recommend this method as a routine test for the early and rapid diagnosis of tuberculosis. 130
    • Summary Summary One-third of the world population is infected with M.tuberculosis and at risk for active disease. Although the lung is the primary site of disease in 80 to 84 % of tuberculosis cases, extra-pulmonary tuberculosis has become more common with the advent of HIV infection. The recent resurgence in tuberculosis worldwide has renewed interest in new methods for accurate and rapid diagnosis. Currently, developing countries rely on acid-fast staining of sputa or cultures of M. tuberculosis in conjunction with assessment of clinical symptoms and radiographic evidence to diagnose TB. Detection by stain and culture lacks sensitivity, particularly in cases of sputum-negative disease, while chest lesions identified by radiograph cannot identify the causal agent. PCR is highly sensitive but expensive and relies on sophisticated equipment and a clean, preferably aseptic, environment. These conditions are often lacking in developing countries. Extrapulmonary TB presents even more problems, as sputum samples are often not available and obtaining specimens from the suspected site of infection often involves highly invasive and expensive procedures. Recently, we have developed an enzyme immunoassay based on monoclonal antibody; dot-ELISA for the simple and rapid detection of a 55-kDa Mycobacterium antigen in serum of pulmonary tuberculosis. In the present study : 1- Western blot analysis revealed that TB-55 mAb reacted against an antigen at an apparent molecular weight of 55 kDa in BCG vaccine, ascites fluid and CSF and serum samples of pulmonary and extra-pulmonary tuberculosis patients but no reaction was observed in serum samples, ascites fluid and CSF of controls. In addition, a high molecular weight target epitope was identified at 82 kDa in BCG vaccine. 131
    • Summary 2- The 55–kDa target antigen was purified from ascites fluid and CSF of extrapulmonary tuberculosis and serum samples of pulmonary and extrapulmonary tuberculosis patients using electroelution from polyacrylamide preparative slab gels. Purified antigen showed a single band in comassie blue stained SDS-PAGE and one peak when analyzed by capillary zone electrophoresis at 11 min. The reactive epitope of the purified antigen was destroyed after treatment with acid and base hydrolysis, mercaptoethanol, protease, and pepsin treatments. The purified antigen was precipitated with 40% TCA, and reconstituted in PBS, pH 7.2. The TB 55 mAb was used as a probe in dot-ELISA. A color dot corresponding to the purified antigen with 55 kDa was observed in TCA reconstituted precipitated fraction but no reaction with TCA soluble fraction was observed. Periodate treatment did not affect the reactivity of the target epitopes of purified antigen. The amino acid analysis of the purified 55- kDa antigen, using high performance liquid chromatograph showed that the 55- kDa of M. tuberculosis antigen consisted of 15 amino acids (leucine, isoleucine, valine, proline, methionine, tyrosine, alanine, glycine, serine, theronine, lysine, arginine, histidine) glutamic and aspartic. The hydrophobic amino acids (leucine, isoleucine, valine, proline, methionine, tyrosine and alanine) represented 24.6% while the hydrophilic amino acids (glycine, serine and theronine) represented 46.4%. Basic amino acids (lysine, arginine and histidine) represented 16.3% and acidic amino acids (glutamic and aspartic) represented 12.7%. So, the 55- kDa antigen is a basic polypeptide chain with a hydrophilic nature. 3- TB-55 mAb antibody was used as a probe in dot-ELISA to detect a target tuberculosis antigen in serum. This is a semi-quantitative assay, requires no sophisticated equipment, rapid (5 minutes) and requires little or no skill to perform without pretreatment of serum sample and the results can be read 132
    • Summary visually without the need of ELISA reader. The result is a colored spot in case of TB antigen detection (i.e. positive test). 4. Serum samples of 506 individuals were included in the present study. They included patients with pulmonary TB (n= 296), patients with extrapulmonary TB (n= 93) as well as sera of patients with respiratory diseases other than TB (n= 69 ) and healthy controls (n= 48). Serum samples of the 389 tuberculosis patients were screened by the dot-ELISA. Of the 389 cases examined, 296 were pulmonary tuberculosis (76%) and 93 cases (24 %) were extra-pulmonary tuberculosis. Patients with extra pulmonary TB were classified according to the location of the infection as follow: peritonitis tuberculosis (27 %), meningitis tuberculosis (24 %), genitourinary tract (20 %), lymph nodes (16 %), potts disease (5 %), arthritis (3 %), sinusitis (3 %), millary (2%). 5. Serum samples of patients with pulmonary TB (n= 296) were tested for circulating 55- kDa using dot-ELISA. Of 296 pulmonary tuberculosis cases, 257 were positive for TB antigen (87 %) and 39 cases (13 %) were negative for TB antigen. Levels of circulating 55-kDa antigen using Dot- ELISA in serum samples of pulmonary patients were negative (13 %), positive with low antigen level (73%) and positive with high antigen level (14 %). The antigen was detected in serum samples of pulmonary tuberculosis patients with sensitivity (87 %), 97 % specificity, 90 % efficiency, positive predictive value (98 %), negative predictive value (74 %), 6. Serum samples of patients with extra pulmonary TB (n= 93) were tested for circulating 55- kDa using dot-ELISA. Of 93 cases, 84 were positive for TB antigen (90 %) and 9 cases (10 %) were negative for TB antigen. The detection rate of extra-pulmonary tuberculosis using dot ELISA were 88 %, 91%, 89%, 86 %, 100 %, 100 %, 100 % and 100 % in TB peritonitis, TB 133
    • Summary meningitis, TB genitourinary tract, TB lymphadenitis, Pott's disease, TB sinusitis, TB arthritis and milliary TB ; respectively. Levels of circulating 55kDa antigen using Dot- ELISA in serum samples of extra-pulmonary patients were negative (10 %), positive with low antigen level (62 %) and positive high antigen level (28 %). The sensitivity, specificity, efficiency, positive predictive value , negative predictive value of the Dot-ELISA for the detection of TB circulating 55-kDa antigen in serum samples of extrapulmonary tuberculosis were 90 %,97 %, 94 % 95 % and 93 %; respectively. 7. Overall levels of circulating 55-kDa antigen using Dot- ELISA in serum samples of pulmonary and extra-pulmonary patients. 48 out of 389 pulmonary and extra-pulmonary tuberculosis (12.3 %) were negative, 273 (70.2 %) were positive with low antigen level and 68 (17.5 %) were positive with high antigen level. The overall advantage of TB-circulating 55-kDa antigen detection by using dot- ELISA in serum samples was evaluated. The antigen was detected in serum samples of tuberculosis patients with sensitivity (88 %), 97 % specificity, 90 % efficiency, positive predictive value (99 %), negative predictive value (70 %). All samples showing false negative results (n= 48) using dot-ELISA were tested using the more sensitive Western blot and 55-kDa antigen was detected in all (100 %) false negative samples. In conclusion, the TB-55 mAb antibody identified 55 kDa antigen in ascites fluid, CSF and serum samples of infected individuals using western blot. The 55-kDa antigen was purified from these samples and partialy characterized as a protein. The dot-ELISA detected the 55 kDa antigen in 90% sera of individuals with extra-pulmonary TB and in 87% sera of individuals with 134
    • Summary pulmonary TB with high degree of specificity (97%) among control individuals. The technical aspects of the dot-ELISA can be performed very simply and the staff of a single laboratory can easily handle large number of serum specimens. The test can be used for the initial diagnosis of extra-pulmonary TB such as peritonitis, meningitis, lymphadenitis, genitourinary tract, potts disease, arthritis, sinusitis and millary TB. According to the recommendations of the World Health Organization, to replace the “gold standard”, bacterial culture, a serological test must possess a sensitivity of over 80% and specificity of over 95%. So detection of TB-circulating 55-kDa antigen using dot- ELISA in serum samples may replace M. tuberculosis culture. 135
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    • ‫اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ‬ ‫اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ‬ ‫اﻟﺪرن )اﻟﺴﻞ( ﻣﻦ أﺧﻄﺮ اﻻﻣﺮاض اﻟﺒﻜﺘﯿﺮﯾﺔ اﻟﺘﻰ ﺗﺼﯿﺐ ﺣﻮاﻟﻰ ﺛﻠﺚ ﺳﻜﺎن اﻟﻌﺎﻟﻢ وﯾﺘﺴﺒﺐ‬ ‫ﺳﻨﻮﯾﺎ ﻓ ﻰ 8 ﻣﻠﯿ ﻮن إﺻ ﺎﺑﺔ ﺟﺪﯾ ﺪة ووﻓ ﺎة 3 ﻣﻠﯿ ﻮن ﺷ ﺨﺺ ﺣﯿ ﺚ ﺗ ﺼﯿﺐ ﺑﻜﺘﯿﺮﯾ ﺎ اﻟ ﺪرن اﻟﺮﺋ ﮫ ﻓ ﻰ‬ ‫08-48% ﻣﻦ اﻟﺤﺎﻻت أو ﺗﻨﺘﻘﻞ اﻟﻰ أﻋﻀﺎء اﻟﺠﺴﻢ اﻻﺧ ﺮى ﻓ ﻰ ﺑ ﺎﻗﻰ اﻟﺤ ﺎﻻت. وﯾﻌﺘﻤ ﺪ ﺗ ﺸﺨﯿﺺ‬ ‫اﻟ ﺪرن اﻟﺮﺋ ﻮى )‪ (pulmonaly tuberculosis‬ﻋﻠ ﻰ اﻟﻔﺤ ﺺ اﻟ ﺴﺮﯾﺮى واﻟﻔﺤﻮﺻ ﺎت اﻟﺘﺸﺨ ﺼﯿﺔ‬ ‫وﻣﻨﮭ ﺎ اﻟﻔﺤ ﺺ اﻟﻤﺠﮭ ﺮى ﻟﻠ ﺒﻠﻐﻢ أو ﻋﻤ ﻞ ﻣﺰرﻋ ﺔ ﻟ ﮫ أو ﺑﻌﻤ ﻞ أﺷ ﻌﺔ ﻋﻠ ﻰ اﻟ ﺼﺪر أو اﻟﻜ ﺸﻒ ﻋ ﻦ‬ ‫اﻟﺤﺎﻣﺾ اﻟﻨﻮوى ﻟﺒﻜﺘﯿﺮﯾﺎ اﻟﺪرن ﺑﺎﺳﺘﺨﺪم ﺗﻔﺎﻋﻞ اﻟﺒﻠﻤ ﺮه اﻟﻤﺘﺴﻠ ﺴﻞ ‪،Polymerase chain reaction‬‬ ‫أﻣﺎ ﺗ ﺸﺨﯿﺺ اﻟ ﺪرن ﺧ ﺎرج اﻟﺮﺋ ﺔ )‪ (Extra-pulmonaly tuberculosis‬ﻓﺎﻧ ﮫ أﻛﺜ ﺮ ﺗﻌﻘﯿ ﺪً ﻣ ﻦ اﻟ ﺪرن‬ ‫ا‬ ‫اﻟﺮﺋﻮى ﻻﻧﮫ ﯾﻌﺘﻤﺪ ﻋﻠﻰ ﻋﯿﻨﺔ ﻣﻦ ﻧﺴﯿﺞ اﻟﻌﻀﻮ اﻟﻤﺼﺎب )‪ (Biopsy‬ﺛﻢ ﻓﺤﺼﮭﺎ ﺑﺎﺛﻮﻟﻮﺟﯿ ﺎ. وﺣ ﺪﯾﺜﺎ‬ ‫ﺗ ﻢ ﺗﻄ ﻮﯾﺮ ﻃﺮﯾﻘ ﺔ ﻣﻨﺎﻋﯿ ﺔ ﺑ ﺴﯿﻄﺔ وﺳ ﮭﻠﺔ وھ ﻰ اﻷﻟﯿ ﺰا اﻟﻨﻘﻄﯿ ﺔ ﺗﻌﺘﻤ ﺪ ﻋﻠ ﻰ أﺳ ﺘﺨﺪام ﺟ ﺴﻢ ﻣ ﻀﺎد‬ ‫أﺣﺎدى اﻟﻨﺴﯿﻠﺔ ﻟﺘﻌﯿﯿﻦ أﺣﺪ أﻧﺘﯿﺠﯿﻨﺎت ﺑﻜﺘﯿﺮﯾﺎ اﻟ ﺪرن ) 55 ﻛﯿﻠﻮداﻟﺘ ﻮن( ﻓ ﻲ ﻋﯿﻨ ﺎت ﺳ ﯿﺮم ﻟﻠﻤﺮﺿ ﻰ‬ ‫ﻣﺼﺎﺑﯿﻦ ﺑﺎﻟﺪرن اﻟﺮﺋﻮي.‬ ‫وﺗﮭ ﺪف ھ ﺬه اﻟﺪراﺳ ﺔ إﻟ ﻰ اﻟﺘﻌ ﺮف ﻋﻠ ﻰ أﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن 55 ﻛﯿﻠﻮداﻟﺘ ﻮن ﻓ ﻲ ﺳ ﻮاﺋﻞ اﻟﺠ ﺴﻢ‬ ‫وﺗﻘﯿ ﯿﻢ ﻛﻔﺎﺋ ﺔ أﺧﺘﺒ ﺎر اﻷﻟﯿ ﺰا اﻟﻨﻘﻄﯿ ﺔ ﻓ ﻰ ﺗﻌﯿ ﯿﻦ ھ ﺬا اﻻﻧﺘﺠ ﯿﻦ ﻓ ﻰ ﻋﯿﻨ ﺎت ﺳ ﯿﺮم ﻟﻤﺮﺿ ﻰ ﻣ ﺼﺎﺑﯿﻦ‬ ‫ﺑﺎﻟﺪرن اﻟﺮﺋﻮى وﺧﺎرج اﻟﺮﺋﺔ.‬ ‫وﻗﺪ اﺷﺘﻤﻠﺖ ھﺬه اﻟﺪراﺳﺔ ﻋﻠﻰ اﻵﺗﻲ:‬ ‫1. ﺗﻢ ﻓﺼﻞ اﻧﺘﯿﺠﯿﻨﺎت ﻟﻘﺎح )‪ (BCG‬وﻋﯿﻨﺎت ﺳ ﯿﺮم اﻟﻤﺮﺿ ﻰ اﻟﻤ ﺼﺎﺑﯿﻦ ﺑﺎﻟ ﺪرن اﻟﺮﺋ ﻮي و ﺑﺎﻟ ﺪرن‬ ‫ﺧ ﺎرج اﻟﺮﺋ ﺔ و ﺳ ﺎﺋﻞ اﻟﻨﺨ ﺎع اﻟ ﺸﻮﻛﻰ )‪ (CSF‬وﺳ ﺎﺋﻞ اﻻﺳﺘ ﺴﻘﺎء )‪ (ascites‬ﺑﺎﺳ ﺘﺨﺪام ﻃﺮﯾﻘ ﺔ‬ ‫اﻟﺒ ﻮﻟﻲ أﻛﺮﯾﻠﻤﯿ ﺪ ﺟ ﻞ اﻟﻜﺘﺮوﻓ ﻮرﯾﺰس )‪ (polyacrylamide gel electrophoresis‬ﺛ ﻢ اﻟﺘﻌ ﺮف‬ ‫ﻋﻠﻰ اﻟﺒﺮوﺗﯿﻨﺎت اﻟﻤﻔﺼﻮﻟﺔ ﺑﺼﺒﻐﮭﺎ ﺑ ﺼﺒﻐﺔ اﻟﻜﻮﻣﺎﺳ ﻰ اﻟﺰرﻗ ﺎء ‪ Coomassie blue‬ﺛ ﻢ اﻟﺘﻌ ﺮف‬ ‫ﻋﻠﻰ اﻷﻧﺘﯿﺠﯿﻦ اﻟﺬي ﯾﺤﻮى اﻟﺠﺰء اﻟﻔﻌﺎل ‪ Reactive epitope‬اﻟﺨﺎص ﺑﺎﻟﺠ ﺴﻢ اﻟﻤ ﻀﺎد أﺣ ﺎدى‬ ‫اﻟﻨ ﺴﯿﻠﺔ‬ ‫‪mAb‬‬ ‫55‬ ‫‪ TB‬ﻓ ﻲ ﺗﻠ ﻚ اﻟﻌﯿﻨ ﺎت ﺑﺎﺳ ﺘﺨﺪام ﻃﺮﯾﻘ ﺔ اﻟ ﺸﻔﻂ اﻟﻤﻨ ﺎﻋﻲ‬‫1‬
    • ‫اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ‬ ‫‪ Immunoblotting technique‬ﺛﻢ ﺗﻌﯿ ﯿﻦ اﻟ ﻮزن اﻟﺠﺰﺋ ﻲ ﻻﻧﺘﯿﺠ ﯿﻦ اﻟ ﺬي ﯾﺤ ﻮى اﻟﺠ ﺰء اﻟﻔﻌ ﺎل‬ ‫اﻟﺨﺎص ﺑﺎﻟﺠﺴﻢ اﻟﻤﻀﺎد ﺑﺎﺳﺘﺨﺪام ﺧﻠﯿﻂ ﻣﻦ ﺑﺮوﺗﯿﻨﺎت ﻗﯿﺎﺳﯿﺔ ﻣﻌﻠﻮﻣﺔ اﻟﻮزن اﻟﺠﺰﺋﻲ وﻗﺪ وﺟ ﺪ‬ ‫أن اﻟﻮزن اﻟﺠﺰﺋﯿﻲ ﻟﮭﺬا اﻷﻧﺘﯿﺠﯿﻦ ھ ﻮ 55 ﻛﯿﻠﻮداﻟﺘ ﻮن ﻓ ﻲ ﺗﻠ ﻚ اﻟﻌﯿﻨ ﺎت ﺑﺎﻹﺿ ﺎﻓﺔ إﻟ ﻰ أﻧﺘﺠ ﯿﻦ‬ ‫أﺧﺮ ذو وزن ﺟﺰﺋﯿﻰ28 ﻛﯿﻠﻮداﻟﺘﻮن ﻓﻲ ﻟﻘﺎح )‪.(BCG‬‬ ‫2- ﺗﻢ ﺗﻨﻘﯿﺔ اﻷﻧﺘﯿﺠﯿﻦ 55 ﻛﯿﻠﻮداﻟﺘﻮن ﻣﻦ ﺳﯿﺮم اﻟﻤﺮﺿ ﻰ اﻟﻤ ﺼﺎﺑﯿﻦ ﺑﺎﻟ ﺪرن اﻟﺮﺋ ﻮي وﻣ ﻦ ﺳ ﯿﺮم‬ ‫اﻟﻤﺮﺿ ﻰ اﻟﻤ ﺼﺎﺑﯿﻦ ﺑﺎﻟ ﺪرن ﺧ ﺎرج اﻟﺮﺋ ﺔ وﻣ ﻦ ﺳ ﺎﺋﻞ اﻟﻨﺨ ﺎع اﻟ ﺸﻮﻛﻰ وﺳ ﺎﺋﻞ اﻻﺳﺘ ﺴﻘﺎء‬ ‫ﺑﺎﺳ ﺘﺨﺪام اﻟﺠ ﻞ اﻟﺘﺤ ﻀﯿﺮي )‪ ( Preparative gel gelelectrophoresis‬ﺛ ﻢ ﺗﻨﻘﯿﺘ ﺔ ﻣ ﻦ اﻟﺠﯿ ﻞ‬ ‫ﺑﻄﺮﯾﻘ ﺔ اﻹزاﺣ ﺔ اﻟﻜﮭﺮﺑﯿ ﺔ )‪ ( Electroelution‬ﺗ ﻢ ﺗﻮﺻ ﯿﻒ ھ ﺬه اﻷﻧﺘﯿﺠﯿﻨ ﺎت ﻟﻤﻌﺮﻓ ﺔ اﻟﻄﺒﯿﻌ ﺔ‬ ‫اﻟﻜﯿﻤﯿﺎﺋﯿ ﺔ ﻟﻠﺠ ﺰء اﻟﻔﻌ ﺎل ‪ Reactive epitope‬ﺑﺎﺳ ﺘﺨﺪام اﻟﺠ ﺴﻢ اﻟﻤ ﻀﺎد أﺣ ﺎدى اﻟﻨ ﺴﯿﻠﺔ‬ ‫‪ TB- 55 mAb‬ﻓﻮﺟﺪ أن ھﺬه اﻷﻧﺘﯿﺠﯿﻨﺎت ﻋﺒ ﺎرة ﻋ ﻦ ﺳﻠ ﺴﻠﺔ ﻣﻔ ﺮده ﻋﺪﯾ ﺪة اﻟﺒﯿﺘﯿ ﺪات وﺗﻌﻄ ﻰ‬ ‫ﻗﻤ‬ ‫ﺔ )‪ (peak‬واﺣ‬ ‫ﺪة ﻋﻨ‬ ‫ﺪ اا دﻗﯿﻘ‬ ‫ﺔ ﻋﻨ‬ ‫ﺪ اﻟﻔ‬ ‫ﺼﻞ ﻋﻠ‬ ‫ﻰ ﺟﮭ‬ ‫ﺎز‬ ‫)‪ (Capillary zone electrophoresis‬و ﯾﺘﺮﺳ ﺐ ﺑﻤ ﺎدة ‪ Tricholoroacetic acid‬ﻻ ﯾﺘ ﺄﺛﺮ‬ ‫ﺑﺎﻷﻛﺴﺪة ﺑﺎﺳﺘﺨﺪام ‪ Periodate‬وﯾﺘﺤﻠﻞ ﺑﺎﻷﻧﺰﯾﻤﺎت اﻟﻤﺤﻠﻠﺔ ﻟﻠﺒﺮوﺗﯿﻨ ﺎت ووﺟ ﺪ أﯾ ﻀﺎ أن ﻓﻌﺎﻟﯿ ﺔ‬ ‫ھ ﺬه اﻷﻧﺘﯿﺠﯿﻨ ﺎت ﻻ ﺗﺘ ﺄﺛﺮ ﺑ ﺎﻟﮭﺠﺮة اﻟﻜﮭﺮﺑﯿ ﺔ )‪ (Electrophoretic migration‬وﻻ ﯾﺘ ﺄﺛﺮ‬ ‫ﺑﺎﻟﻌﻮاﻣ ﻞ اﻟﻤﺨﺘﺰﻟ ﺔ ﻣﺜ ﻞ )‪ Sodium dodecyl sulfate‬أو ‪ (Mercaptoethanol‬ﻣﻤ ﺎ ﯾ ﺪل ﻋﻠ ﻰ‬ ‫اﻟﺜﺒﺎت اﻟﻌﺎﻟﻲ ﻟﻠﺠﺰء اﻟﻔﻌﺎل ھﺬه اﻷﻧﺘﯿﺠﯿﻨﺎت. ﺗﻢ ﻋﻤﻞ ﺗﺤﻠﯿﻞ اﻷﺣﻤ ﺎض اﻷﻣﯿﻨﯿ ﺔ اﻟﻤﻜﻮﻧ ﺔ ﻟﻌﯿﻨ ﺔ‬ ‫ﻣ ﻦ اﻷﻧﺘﯿﺠﯿﻨ ﺎت ﺑﺎﺳ ﺘﺨﺪام ﺟﮭ ﺎز اﻟﺘﺤﻠﯿ ﻞ اﻟﻜﺮوﻣ ﺎﺗﻮﺟﺮاﻓﻰ اﻟ ﺴﺎﺋﻞ ﻋ ﺎﻟﻰ اﻟﻜﻔ ﺎءة‬ ‫‪ High performance liquid chromatograph‬ﻓﻮﺟ ﺪ أﻧ ﮫ ﯾﺘﻜ ﻮن ﻣ ﻦ 51 ﺣﻤ ﺾ أﻣﯿﻨ ﻰ ﻧ ﺴﺒﮫ‬ ‫اﻷﺣﻤ ﺎض اﻷﻣﯿﻨﯿ ﺔ اﻟﻜﺎرھ ﺔ ﻟﻠﻤ ﺎء ﺑ ﮫ 6.42% وﻧ ﺴﺒﮫ اﻷﺣﻤ ﺎض اﻷﻣﯿﻨﯿ ﺔ اﻟﻤﺤﺒ ﺔ ﻟﻠﻤ ﺎء ھ ﻰ‬ ‫4.64% وﻧﺴﺒﺔ اﻷﺣﻤﺎض اﻷﻣﯿﻨﯿﺔ ذات اﻟﺨﻮاص اﻟﻘﺎﻋﺪﯾﺔ 3.61% وﻧﺴﺒﺔ اﻷﺣﻤﺎض اﻷﻣﯿﻨﯿ ﺔ‬ ‫ذات اﻟﺨﻮاص اﻟﺤﻤﻀﯿﺔ 7.21%.‬ ‫2‬
    • ‫اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ‬ ‫3- ﺗﻢ اﺳﺘﺨﺪام اﻟﺠﺴﻢ اﻟﻤﻀﺎد أﺣﺎدى اﻟﻨ ﺴﯿﻠﺔ ‪ TB- 55 mAb‬ﻟﻠﻜ ﺸﻒ ﻋ ﻦ اﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار‬ ‫55 ﻛﯿﻠﻮداﻟﺘ ﻮن ﻓ ﻲ اﻟ ﺴﯿﺮم ﺑﻄﺮﯾﻘ ﺔ اﻹﻟﯿ ﺰا اﻟﻨﻘﻄﯿ ﺔ )‪ (dot-ELISA‬وھ ﻮ اﺧﺘﺒ ﺎر‬ ‫)‪ (Semi-quantitative‬ﺣﯿﺚ ﯾﻈﮭﺮ ﻟ ﻮن ﺑﻨﻔ ﺴﺠﻲ ﻓ ﻲ ﻋﯿﻨ ﺔ اﻟ ﺸﺨﺺ اﻟﻤ ﺼﺎب ﺑﺒﻜﺘﯿﺮﯾ ﺎ اﻟ ﺪرن‬ ‫وﻻ ﯾﻈﮭﺮ ﻟﻮن ﻓﻲ ﻋﯿﻨﺔ اﻟﺸﺨﺺ اﻟﻐﯿﺮ ﻣﺼﺎب وﺗﺨﺘﻠﻒ درﺟ ﺔ اﻟﻠ ﻮن ﻓ ﻲ اﻟﻌﯿﻨ ﺔ اﻟﻤﻮﺟﺒ ﺔ ﻣ ﻦ‬ ‫اﻟﺪرﺟ ﺔ اﻟ ﻀﻌﯿﻔﺔ )+1،+2( إﻟ ﻰ اﻟﺪرﺟ ﺔ اﻟﻘﻮﯾ ﺔ )+3،+4( ﺣ ﺴﺐ ﻣ ﺴﺘﻮى اﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن‬ ‫اﻟ ﺪوار 55 ﻛﯿﻠﻮداﻟﺘ ﻮن ﻓ ﻲ اﻟ ﺴﯿﺮم ﺣﯿ ﺚ ﺗ ﻢ أﺳ ﺘﺨﺪام 605 ﺣﺎﻟ ﺔ ﻣ ﻨﮭﻢ 983 ﺣﺎﻟ ﺔ ﻣ ﺼﺎﺑﺔ‬ ‫ﺑﺎﻟ ﺪرن و711 ﺣﺎﻟ ﺔ ﻣ ﻦ ﻣﺮﺿ ﻰ اﻣ ﺮاض ﺗﻨﻔ ﺴﯿﺔ ﺻ ﺪرﯾﺔ ﻏﯿ ﺮ اﻟ ﺪرن وأﺻ ﺤﺎء ﻛﻤﺠﻤﻮﻋ ﺔ‬ ‫ﺿ ﺎﺑﻄﺔ، 983 ﻋﯿﻨ ﮫ ﺳ ﯿﺮم ﻟﺤ ﺎﻻت ﻣ ﺼﺎﺑﺔ ﺑﺎﻟ ﺪرن ﻣ ﻨﮭﻢ 692 ﺣﺎﻟ ﺔ ﻣ ﺼﺎﺑﺔ ﺑﺎﻟ ﺪرن اﻟﺮﺋ ﻮى‬ ‫)67%( ، 39 ﺣﺎﻟﺔ )42%( ﻣ ﺼﺎﺑﯿﻦ ﺑﺎﻟ ﺪرن ﺧ ﺎرج اﻟﺮﺋ ﮫ اﻟ ﺬﯾﻦ ﺗ ﻢ ﺗﻘ ﺴﯿﻤﮭﻢ ﺣ ﺴﺐ ﻣﻮﺿ ﻊ‬ ‫اﻻﺻﺎﺑﺔ اﻟﻰ 52 ﺣﺎﻟﺔ درن ﺑﻄﻨ ﻰ ﺑﻨ ﺴﺒﺔ )72%( ، 22 ﺣﺎﻟ ﮫ ﻣ ﺼﺎﺑﺔ ﺑﺎﻟ ﺪرن اﻟ ﺴﺤﺎﺋﻰ ﺑﻨ ﺴﺒﺔ‬ ‫)42%( ، 91 ﺣﺎﻟ ﺔ ﻣ ﺼﺎﺑﺔ ﺑﺎﻟ ﺪرن ﻓ ﻰ اﻟﻘﻨ ﺎة اﻟﺒﻮﻟﯿ ﺔ اﻟﺘﻨﺎﺳ ﻠﯿﺔ ﺑﻨ ﺴﺒﺔ )02%( ، 91 ﺣﺎﻟ ﮫ‬ ‫ﻣﺼﺎﺑﺔ ﺑﺎﻟﺪرن ﻓ ﻰ اﻟﻐ ﺪد اﻟﯿﻤﻔﺎوﯾ ﺔ ﺑﻨ ﺴﺒﺔ )61%( ، 5 ﺣ ﺎﻻت ﻣ ﺼﺎﺑﺔ ﺑﻤ ﺮض ﺑ ﻮﺗﺲ ﺑﻨ ﺴﺒﺔ‬ ‫)5%( ، 3 ﺣ ﺎﻻت ﻣ ﺼﺎﺑﺔ ﺑ ﺪرن ﻓ ﻰ اﻟﻤﻔﺎﺻ ﻞ ﺑﻨ ﺴﺔ )3%( و 3 ﺣ ﺎﻻت ﻣ ﺼﺎﺑﺔ ﺑ ﺪرن ﻓ ﻰ‬ ‫اﻟﺠﯿﻮب اﻷﻧﻔﯿﺔ ﺑﻨﺴﺒﺔ )3%( وﺣ ﺎﻟﺘﯿﻦ ﻣ ﺼﺎﺑﺘﯿﻦ ﺑ ﺪرن ﻓ ﻰ ﻣﻮاﺿ ﻊ ﻣﺨﺘﻠﻔ ﺔ ﻣ ﻦ اﻟﺠ ﺴﻢ ﺑﻨ ﺴﺒﺔ‬ ‫)2%( .‬ ‫ﻓﺎوﺿﺤﺖ اﻟﻨﺘﺎﺋﺞ أن اﻧﺘﯿﺠﯿﻦ اﻟﺪرن اﻟ ﺪوار 55 ﻛﯿﻠﻮداﻟﺘ ﻮن ﯾﻮﺟ ﺪ ﻓ ﻰ 752 ﺣﺎﻟ ﺔ ﻣ ﻦ 692‬ ‫ﺣﺎﻟ ﮫ ﻣ ﺼﺎﺑﺔ ﺑﺎﻟ ﺪرن اﻟﺮﺋ ﻮى ﺑﻨ ﺴﺒﺔ )78%( و 93 ﺣﺎﻟ ﺔ ﻣ ﺼﺎﺑﺔ ﺑﺎﻟ ﺪرن اﻟﺮﺋ ﻮى ﻻ ﯾﻮﺟ ﺪ‬ ‫اﻧﺘﯿﺠﯿﻦ اﻟﺪرن اﻟﺪوار 55 ﻛﯿﻠﻮداﻟﺘﻮن ﺑﻨﺴﺒﺔ )31%(. وﻟﺘﻘﯿﯿﻢ ﻛﻔﺎﺋﺔ أﺧﺘﺒﺎر اﻷﻟﯿﺰا اﻟﻨﻘﻄﯿﺔ ﻓﻰ‬ ‫ﺗﻌﯿ ﯿﻦ اﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار55 ﻛﯿﻠﻮداﻟﺘ ﻮن ﻓ ﻰ ﻋﯿﻨ ﺎت ﺳ ﯿﺮم ﻟﻤﺮﺿ ﻰ ﻣ ﺼﺎﺑﯿﻦ ﻣ ﺼﺎﺑﯿﻦ‬ ‫ﺑﺎﻟﺪرن اﻟﺮﺋﻮى ﻓﺄوﺿﺤﺖ اﻟﻨﺘﺎﺋﺞ أن 752 ﺣﺎﻟﺔ ﯾﻮﺟﺪ ﺑﮭﺎ أﻧﺘﺠﯿﻦ اﻟﺪرن اﻟﺪوار ﻣﻦ 692 ﺣﺎﻟ ﺔ‬ ‫ﻣﺼﺎﺑﮫ ﺑﺎﻟﺪرن اﻟﺮﺋﻮى )ﺣﺴﺎﺳﯿﺔ 78%( ، 311 ﺣﺎﻟﺔ ﻣﻦ 711 ﺣﺎﻟﺔ ﻣﻦ اﻟﻤﺠﻤﻮﻋﺔ اﻟ ﻀﺎﺑﻄﺔ‬ ‫)ﺣﺎﻻت ﻣﺼﺎﺑﺔ ﺑﺄﻣﺮاض ﺻﺪرﯾﺔ ﻏﯿﺮ اﻟﺪرن وأﺷﺨﺎص ﻃﺒﯿﻌﯿﻦ( ﻻ ﯾﻮﺟ ﺪ ﺑﮭ ﺎ أﻧﺘﺠ ﯿﻦ اﻟ ﺪرن‬ ‫3‬
    • ‫اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ‬ ‫)ﺧﺼﻮﺻﯿﺔ 79%( وﻛﻔﺎﺋﺔ 09% وﻗﯿﻤ ﺔ ﺗﻨﺒﺆﯾ ﺔ ﻣﻮﺟﺒ ﺔ 89 % وﻗﯿﻤ ﺔ ﺗﻨﺒﺆﯾ ﺔ ﺳ ﺎﻟﺒﺔ 47 % .‬ ‫ﺗﻢ ﺗﺤﺪﯾﺪ ﻣﺴﺘﻮى اﻧﺘﯿﺠﯿﻦ اﻟ ﺪرن ﻓﻜﺎﻧ ﺖ ﻧﺘ ﺎﺋﺞ 692 ﺣﺎﻟ ﺔ ﻣ ﺼﺎﺑﺔ ﺑﺎﻟ ﺪرن اﻟﺮﺋ ﻮى ﻛ ﺎﻻﺗﻰ:93‬ ‫ﺣﺎﻟﺔ ﻻﯾﻮﺟﺪ ﺑﮭﺎ اﻧﺘﯿﺠﯿﻦ اﻟﺪرن اﻟﺪوار ﺑﻨﺴﺒﺔ )31%( و512 ﺣﺎﻟﺔ ﯾﻮﺟﺪ ﺑﮭﺎ ﻣ ﺴﺘﻮى ﺿ ﻌﯿﻒ‬ ‫ﻻﻧﺘﯿﺠﯿﻦ اﻟﺪرن اﻟﺪوار ﺑﻨﺴﺒﺔ )37%( ﺑﺎﻷﺿﺎﻓﺔ اﻟﻰ 24 ﺣﺎﻟﺔ ﯾﻮﺟﺪ ﺑﮭﺎ ﻣﺴﺘﻮى ﻗﻮى ﻻﻧﺘﯿﺠﯿﻦ‬ ‫اﻟﺪرن اﻟﺪوار ﺑﻨﺴﺒﺔ )41%(‬ ‫4. ﺗ ﻢ اﺳ ﺘﺨﺪام اﻻﻟﯿ ﺰا اﻟﻨﻘﻄﯿ ﺔ )‪ (dot ELISA‬ﻛﻄﺮﯾﻘ ﺔ ﺳ ﮭﻠﺔ وﺑ ﺴﯿﻄﺔ ﻟﻠﻜ ﺸﻒ ﻋ ﻦ اﻧﺘﯿﺠ ﯿﻦ ﻓ ﻰ‬ ‫ﻋﯿﻨﺎت ﺳﯿﺮم ﺣﺎﻻت ﻣﺼﺎﺑﺔ ﺑﺎﻟﺪرن ﺧﺎرج اﻟﺮﺋ ﺔ ﻓﺎوﺿ ﺤﺖ اﻟﻨﺘ ﺎﺋﺞ أن اﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار‬ ‫55 ﻛﯿﻠﻮداﻟﺘﻮن ﯾﻮﺟﺪ ﻓﻰ 48 ﺣﺎﻟﺔ ﻣﻦ 39 ﺣﺎﻟﮫ ﻣﺼﺎﺑﺔ ﺑﺎﻟﺪرن ﺧ ﺎرج اﻟﺮﺋ ﺔ ﺑﻨ ﺴﺒﺔ )09%(‬ ‫و 9 ﺣﺎﻻت ﻣﺼﺎﺑﺔ ﺑﺎﻟﺪرن ﺧﺎرج اﻟﺮﺋﺔ ﻻ ﯾﻮﺟﺪ اﻧﺘﯿﺠﯿﻦ اﻟ ﺪرن اﻟ ﺪوار 55 ﻛﯿﻠﻮداﻟﺘ ﻮن ﺑﻨ ﺴﺒﺔ‬ ‫)01%(. وﻟﺘﻘﯿﯿﻢ ﻛﻔﺎﺋﺔ أﺧﺘﺒﺎر اﻷﻟﯿﺰا اﻟﻨﻘﻄﯿﺔ ﻓﻰ ﺗﻌﯿﯿﻦ اﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار 55 ﻛﯿﻠﻮداﻟﺘ ﻮن‬ ‫ﻓﻰ ﻋﯿﻨﺎت ﺳﯿﺮم ﻟﻤﺮﺿﻰ ﻣﺼﺎﺑﯿﻦ ﺑﺎﻟﺪرن ﺧ ﺎرج اﻟﺮﺋ ﺔ ﻓﺄوﺿ ﺤﺖ اﻟﻨﺘ ﺎﺋﺞ أن 48 ﺣﺎﻟ ﺔ ﯾﻮﺟ ﺪ‬ ‫ﺑﮭﺎ أﻧﺘﺠﯿﻦ اﻟﺪرن اﻟﺪوار ﻣﻦ 39 ﺣﺎﻟﺔ ﻣﺼﺎﺑﮫ ﺑﺎﻟ ﺪرن اﻟﺮﺋ ﻮى )ﺣ ﺴﺎﺳﯿﺔ 09%( ، 311 ﺣﺎﻟ ﺔ‬ ‫ﻣ ﻦ 711 ﺣﺎﻟ ﺔ ﻣ ﻦ اﻟﻤﺠﻤﻮﻋ ﺔ اﻟ ﻀﺎﺑﻄﺔ )ﺣ ﺎﻻت ﻣ ﺼﺎﺑﺔ ﺑ ﺄﻣﺮاض ﺻ ﺪرﯾﺔ ﻏﯿ ﺮ اﻟ ﺪرن‬ ‫وأﺷ ﺨﺎص ﻃﺒﯿﻌ ﯿﻦ( ﻻ ﯾﻮﺟ ﺪ ﺑﮭ ﺎ أﻧﺘﺠ ﯿﻦ اﻟ ﺪرن )ﺧ ﺼﻮﺻﯿﺔ 79%( وﻛﻔﺎﺋ ﺔ 49% وﻗﯿﻤ ﺔ‬ ‫ﺗﻨﺒﺆﯾ ﺔ ﻣﻮﺟﺒ ﺔ 59 % وﻗﯿﻤ ﺔ ﺗﻨﺒﺆﯾ ﺔ ﺳ ﺎﻟﺒﺔ 39 % . ﺗ ﻢ ﺗﺤﺪﯾ ﺪ ﻣ ﺴﺘﻮى اﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن ﻓﻜﺎﻧ ﺖ‬ ‫ﻧﺘ ﺎﺋﺞ 39 ﺣﺎﻟ ﺔ ﻣ ﺼﺎﺑﺔ ﺑﺎﻟ ﺪرن ﺧ ﺎرج اﻟﺮﺋ ﺔ ﻛ ﺎﻻﺗﻰ:9 ﺣ ﺎﻻت ﻻ ﯾﻮﺟ ﺪ ﺑﮭ ﺎ اﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن‬ ‫اﻟ ﺪوار ﺑﻨ ﺴﺒﺔ )01%( و85 ﺣﺎﻟ ﺔ ﯾﻮﺟ ﺪ ﺑﮭ ﺎ ﻣ ﺴﺘﻮى ﺿ ﻌﯿﻒ ﻻﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار ﺑﻨ ﺴﺒﺔ‬ ‫)26%( ﺑﺎﻷﺿ ﺎﻓﺔ اﻟ ﻰ 62 ﺣﺎﻟ ﺔ ﯾﻮﺟ ﺪ ﺑﮭ ﺎ ﻣ ﺴﺘﻮى ﻗ ﻮى ﻻﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار ﺑﻨ ﺴﺒﺔ‬ ‫)82%(.‬ ‫5. ﺗ ﻢ ﺗﺤﺪﯾ ﺪ اﻟﻤ ﺴﺘﻮى اﻟﻜﻠ ﻰ ﻷﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن ﻟﻤﺮﺿ ﻰ ﻣ ﺼﺎﺑﯿﻦ ﺑﺎﻟ ﺪرن اﻟﺮﺋ ﻮى وﺧ ﺎرج اﻟﺮﺋ ﺔ‬ ‫ﻓﻜﺎﻧﺖ ﻧﺘﺎﺋﺞ 983 ﺣﺎﻟ ﺔ ﻣ ﺼﺎﺑﺔ ﺑﺎﻟ ﺪرن ﻛ ﺎﻻﺗﻰ:84 ﺣﺎﻟ ﺔ ﻻ ﯾﻮﺟ ﺪ ﺑﮭ ﺎ اﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار‬ ‫ﺑﻨ ﺴﺒﺔ )3.21%( و372 ﺣﺎﻟ ﺔ ﯾﻮﺟ ﺪ ﺑﮭ ﺎ ﻣ ﺴﺘﻮى ﺿ ﻌﯿﻒ ﻻﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار ﺑﻨ ﺴﺒﺔ‬ ‫4‬
    • ‫اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ‬ ‫)2.07%( ﺑﺎﻷﺿ ﺎﻓﺔ اﻟ ﻰ 86 ﺣﺎﻟ ﺔ ﯾﻮﺟ ﺪ ﺑﮭ ﺎ ﻣ ﺴﺘﻮى ﻗ ﻮى ﻻﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار ﺑﻨ ﺴﺒﺔ‬ ‫)5.71%(. وﻟﺘﻘﯿﯿﻢ ﻛﻔﺎﺋﺔ أﺧﺘﺒﺎر اﻷﻟﯿﺰا اﻟﻨﻘﻄﯿﺔ اﻟﻜﻠﯿﺔ ﻓﻰ ﺗﻌﯿﯿﻦ اﻧﺘﯿﺠﯿﻦ اﻟﺪرن اﻟﺪوار‬ ‫55‬ ‫ﻛﯿﻠﻮداﻟﺘ ﻮن ﻓ ﻰ ﻋﯿﻨ ﺎت ﺳ ﯿﺮم ﻟﻤﺮﺿ ﻰ ﻣ ﺼﺎﺑﯿﻦ ﺑﺎﻟ ﺪرن اﻟﺮﺋ ﻮى وﺧ ﺎرج اﻟﺮﺋ ﺔ ﻓﺄوﺿ ﺤﺖ‬ ‫اﻟﻨﺘ ﺎﺋﺞ أن اﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار ﯾﻮﺟ ﺪ ﻓ ﻰ 143 ﺣﺎﻟ ﺔ ﻣ ﻦ 983 ﺣﺎﻟ ﺔ ﻣ ﺼﺎﺑﺔ ﺑﺎﻟ ﺪرن‬ ‫)ﺣﺴﺎﺳﯿﺔ 88 %( و 4 ﺣ ﺎﻻت ﻓﻘ ﻂ ﻣ ﻦ 711 ﺣﺎﻟ ﺔ ﻣ ﻦ اﻟﻌﯿﻨ ﺎت اﻟ ﻀﺎﺑﻄﺔ )ﻣﺮﺿ ﻰ اﻣ ﺮاض‬ ‫ﺗﻨﻔ ﺴﯿﺔ ﻏﯿ ﺮ اﻟ ﺪرن وأﺻ ﺤﺎء( ﯾﻮﺟ ﺪ ﺑﮭ ﺎ اﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار )ﺧ ﺼﻮﺻﯿﺔ 79%( وﻛﻔﺎﺋ ﺔ‬ ‫أﺧﺘﺒﺎر اﻧﺘﯿﺠﯿﻦ اﻟﺪرن اﻟﺪوار )09%( وﻛﺎﻧﺖ اﻟﻘﯿﻤﺔ اﻟﺘﻨﺒﺆﯾﺔ اﻟﻤﻮﺟﺒ ﺔ ﻻﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار‬ ‫99 % و اﻟﻘﯿﻤﺔ اﻟﺘﻨﺒﺆﯾﺔ اﻟﺴﺎﻟﺒﺔ ﻻﻧﺘﯿﺠﯿﻦ اﻟﺪرن اﻟﺪوار 37 %.‬ ‫واﻟﺨﻼﺻﺔ:‬ ‫ﺗ ﻢ ﺗﻌﺮﯾ ﻒ و ﺗﻨﻘﯿ ﺔ وﺗﻮﺻ ﯿﻒ أﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار 55 ﻛﯿﻠﻮداﻟﺘ ﻮن ﻓ ﻰ ﻋﯿﻨ ﺎت اﻟ ﺴﯿﺮم‬ ‫وﺳ ﺎﺋﻞ اﻟﻨﺨ ﺎع اﻟ ﺸﻮﻛﻰ وﺳ ﺎﺋﻞ اﻷﺳﺘ ﺴﻘﺎء ﻟﻠﻤﺮﺿ ﻰ اﻟﻤ ﺼﺎﺑﯿﻦ ﺑﺎﻟ ﺪرن وﺑﺎﻟﺘ ﺎﻟﻰ ﯾﻤﻜ ﻦ اﺳ ﺘﺨﺪاﻣﮫ‬ ‫ﻛﻜﺎﺷﻒ ﻓﻰ اﻟﺘﺸﺨﯿﺺ اﻟﻤﻨﺎﻋﻰ ﻟﻠﺪرن. ﻃﺮﯾﻘﺔ اﻹﻟﯿﺰا اﻟﻨﻘﻄﯿﺔ ﻟﻠﻜ ﺸﻒ ﻋ ﻦ اﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن اﻟ ﺪوار‬ ‫ﻓﻰ ﻋﯿﻨﺎت ﺳﯿﺮم ﻟﻤﺮﺿﻰ ﻣﺼﺎﺑﯿﻦ ﺑﺎﻟ ﺪرن اﻟﺮﺋ ﻮى وﺧ ﺎرج اﻟﺮﺋ ﺔ ﻃﺮﯾﻘ ﺔ ذات درﺟ ﺎت ﻋﺎﻟﯿ ﺔ ﻣ ﻦ‬ ‫اﻟﺤﺴﺎﺳﯿﺔ 88% واﻟﺨ ﺼﻮﺻﯿﺔ 59% وﯾﻤﻜﻨﮭ ﺎ ﺗ ﺸﺨﯿﺺ أﻧ ﻮاع ﻣﺨﺘﻠﻔ ﺔ ﻣ ﻦ اﻟ ﺪرن ﺧ ﺎرج اﻟﺮﺋ ﺔ‬ ‫ﻣﺜﻞ اﻟﺪرن اﻟﺒﻄﻨﻰ و اﻟﺪرن اﻟﺴﺤﺎﺋﻰ ودرن اﻟﻘﻨﺎة اﻟﺒﻮﻟﯿﺔ اﻟﺘﻨﺎﺳ ﻠﯿﺔ ودرن اﻟﻐ ﺪد اﻟﯿﻤﻔﺎوﯾ ﺔ و ﻣ ﺮض‬ ‫ﺑﻮﺗﺲ ودرن اﻟﻤﻔﺎﺻﻞ ودرن اﻟﺠﯿﻮب اﻷﻧﻔﯿﺔ ودرن ﻓﻰ ﻣﻮاﺿﻊ ﻣﺨﺘﻠﻔﺔ ﻣ ﻦ اﻟﺠ ﺴﻢ ﺑﺎﻷﺿ ﺎﻓﺔ اﻟ ﻰ‬ ‫أﻧﮭﺎ ﺳﺮﯾﻌﺔ وﻏﯿﺮ ﻣﻜﻠﻔﺔ وﻻ ﺗﺤﺘﺎج إﻟ ﻰ أﺟﮭ ﺰة ﻣﻌﻘ ﺪة . وﻃﺒﻘ ﺎ ﻟﺘﻮﺻ ﯿﺎت ﻣﻨﻈﻤ ﺔ اﻟ ﺼﺤﺔ اﻟﻌﺎﻟﻤﯿ ﺔ‬ ‫ﻓﺎن اﻷﺧﺘﺒﺎر اﻟ ﺬى ﯾﻤﻜ ﻦ أن ﯾ ﺴﺘﺒﺪل اﻟﻄﺮﯾﻘ ﺔ اﻟﻘﯿﺎﺳ ﯿﺔ ﻟﺘ ﺸﺨﯿﺺ اﻟ ﺪرن )زراﻋ ﺔ ﺑﻜﺘﯿﺮﯾ ﺎ اﻟ ﺪرن(‬ ‫ﻻﺑﺪ أن ﺗﻜﻮن درﺟ ﺔ ﺣﺎﺳ ﯿﺘﺔ 08 % وﺧ ﺼﻮﺻﯿﺘﺔ 59 % وﻟ ﺬﻟﻚ ﯾﻨ ﺼﺢ ﺑﺎﺳ ﺘﺨﺪام ﻃﺮﯾﻘ ﺔ اﻹﻟﯿ ﺰا‬ ‫اﻟﻨﻘﻄﯿﺔ ﻓﻰ اﻟﻜﺸﻒ اﻟﻤﺒﻜﺮ ﻋﻦ ﻣﺮﺿﻰ اﻟﺪرن.‬ ‫5‬
    • ‫اﻟﻤﺴﺘﺨﻠﺺ‬ ‫اﻻﺳﻢ : ﻣﺤﻤﺪ ﻣﺼﻄﻔﻰ ﻋﻤﺮان‬ ‫ﻋﻨﻮان اﻟﺮﺳﺎﻟﺔ : دراﺳﺎت ﻛﯿﻤﯿﺎﺋﯿﺔ ﺣﯿﻮﯾﺔ ﻋﻠﻰ أﺣﺪ أﻧﺘﯿﺠﯿﻨﺎت ﺑﻜﺘﯿﺮﯾﺎ اﻟﺘﺪرن اﻟﺮﺋﻮي‬ ‫اﻟﺪرﺟﺔ: دﻛﺘﻮراﻟﻔﻠﺴﻔﺔ ﻓﻰ اﻟﻌﻠﻮم )ﻛﯿﻤﯿﺎء ﺣﯿﻮﯾﺔ(‬ ‫ﻣﻠﺨ ﺺ اﻟﺒﺤ ﺚ: اﻟﺘﻌ ﺮف ﻋﻠ ﻰ أﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن ﺧﻄ ﻮه ھﺎﻣ ﺔ ﻧﺤ ﻮ ﺗﺸﺨﯿ ﺼﮫ اﻟ ﺪﻗﯿﻖ وﻓ ﻰ ھ ﺬه‬ ‫اﻟﺪراﺳ ﺔ ﺗ ﻢ اﻟﺘﻌ ﺮف ﻋﻠ ﻰ أﻧﺘﯿﺠ ﯿﻦ اﻟ ﺪرن ﻓ ﻲ ﻋﯿﻨ ﺎت اﻟ ﺴﯿﺮم وﺳ ﺎﺋﻞ اﻻﺳﺘ ﺴﻘﺎء وﺳ ﺎﺋﻞ اﻟﻨﺨ ﺎع‬ ‫اﻟﺸﻮﻛﻰ ﺑﺎﺳﺘﺨﺪام ﺟﺴﻢ ﻣﻀﺎد أﺣ ﺎدى اﻟﻨ ﺴﯿﻠﺔ وﻃﺮﯾﻘ ﺔ اﻟ ﺸﻔﻂ اﻟﻤﻨ ﺎﻋﻲ ﺛ ﻢ ﺗﻨﻘﯿﺘ ﮫ وﺗﻮﺻ ﯿﻔﮫ ﺟﺰﺋﯿ ﺎ‬ ‫ﻛﺒﺮوﺗﯿﻦ ﻟﮫ وزن ﺟﺰﯾﺌﻲ 55 ﻛﯿﻠﻮ داﻟﺘﻮن. ﺗﻢ اﺳﺘﺨﺪام اﻟﯿﺰا اﻟﻨﻘﻄﯿﺔ ﻛﻄﺮﯾﻘﺔ ﻣﻨﺎﻋﯿﺔ ﺳ ﮭﻠﺔ وﺑ ﺴﯿﻄﺔ‬ ‫ﻟﺘﻌﯿﯿﻦ أﻧﺘﯿﺠﯿﻦ اﻟﺪرن ﻓﻲ ﻋﯿﻨ ﺎت اﻟ ﺴﯿﺮم ﺣﯿ ﺚ ﺛﺒ ﺖ وﺟ ﻮده ﻓ ﻰ 09 % ﻣ ﻦ ﺣ ﺎﻻت اﻟ ﺪرن ﺧ ﺎرج‬ ‫اﻟﺮﺋﺔ وﻓﻰ 78 % ﻣﻦ ﺣﺎﻻت اﻟ ﺪرن اﻟﺮﺋ ﻮي وﻗ ﺪ أﻇﮭ ﺮت ھ ﺬه اﻟﻄﺮﯾﻘ ﺔ درﺟ ﺔ ﺧ ﺼﻮﺻﯿﺔ ﻋﺎﻟﯿ ﺔ‬ ‫79 % ﻓﻲ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻀﺎﺑﻄﺔ. اﻟﺨﻼﺻﺔ: ﺗﻢ ﺗﻌﺮﯾﻒ وﺗﻮﺻﯿﻒ ﺟﺰﺋﯿﻲ ﻷﻧﺘﯿﺠﯿﻦ اﻟﺪرن و ﺗﻌﯿﯿﻨﮫ‬ ‫ﻓ ﻲ اﻟ ﺴﯿﺮم ﺑﺎﺳ ﺘﺨﺪام ﻃﺮﯾﻘ ﺔ اﻟﯿ ﺰا اﻟﻨﻘﻄﯿ ﺔ ذات اﻟﺤ ﺴﺎﺳﯿﺔ واﻟﺨ ﺼﻮﺻﯿﺔ اﻟﻌﺎﻟﯿ ﺔ ﻓ ﻲ اﻟﺘ ﺸﺨﯿﺺ‬ ‫اﻟﺮوﺗﯿﻨﻲ وﻟﺘﺪﻋﯿﻢ اﻟﺘﺸﺨﯿﺺ اﻻﻛﻠﯿﻨﻜﻰ ﻟﻠﺪرن.‬ ‫اﻟﻜﻠﻤﺎت اﻟﺪاﻟﮫ: اﻟﺪرن ، اﻟﺘﺸﺨﯿﺺ، أﻧﺘﯿﺠﯿﻦ ، 55 ﻛﯿﻠﻮ داﻟﺘﻮن، ﺳﯿﺮم‬ ‫ﺗﻮﻗﯿﻊ اﻟﺴﺎدة اﻟﻤﺸﺮﻓﻮن‬ ‫1- ا.د ﺳﻨﺎء ﻋﺜﻤﺎن ﻋﺒﺪ اﷲ‬ ‫...............................................................‬ ‫2- ا.د ﻋﺒﺪ اﻟﻔﺘﺎح ﻣﺤﻤﺪ ﻋﻄﺎ اﷲ‬ ‫...............................................................‬ ‫3- د.ﻋﻤﺮو ﺳﻌﺪ ﻣﺤﻤﺪ‬ ‫...............................................................‬ ‫ﯾﻌﺘﻤﺪ ،،،،‬ ‫أ.د / رﻓﻌﺖ ﺣﺴﻦ ھﻼل‬ ‫رﺋﯿﺲ ﻣﺠﻠﺲ ﻗﺴﻢ اﻟﻜﯿﻤﯿﺎء‬ ‫ﻛﻠﯿﺔ اﻟﻌﻠﻮم –ﺟﺎﻣﻌﺔ اﻟﻘﺎھﺮة‬
    • ‫دراﺳﺎت ﻛﯿﻤﯿﺎﺋﯿﺔ ﺣﯿﻮﯾﺔ ﻋﻠﻰ أﺣﺪ أﻧﺘﯿﺠﯿﻨﺎت‬ ‫ﺑﻜﺘﯿﺮﯾﺎ اﻟﺘﺪرن اﻟﺮﺋﻮي‬ ‫رﺳﺎﻟﺔ ﻣﻘﺪﻣﺔ‬ ‫ﻟﻠﺤﺼﻭل ﻋﻠﻰ ﺩﺭﺠﺔ ﺩﻜﺘﻭﺭ ﻓﻠﺴﻔﺔ ﺍﻟﻌﻠﻭﻡ ﻓﻲ ﺍﻟﻜﻴﻤﻴﺎﺀ ﺍﻟﺤﻴﻭﻴﺔ‬ ‫ﻣﻦ اﻟﻄﺎﻟﺐ‬ ‫ﻣﺤﻤﺪ ﻣﺼﻄﻔﻰ ﻋﻤﺮان‬ ‫ﻣﺮﻛﺰ أﺑﺤﺎث اﻟﺘﻜﻨﻮﻟﻮﺟﯿﺎ اﻟﺤﯿﻮﯾﺔ – دﻣﯿﺎط اﻟﺠﺪﯾﺪة‬ ‫ﻗﺴﻢ اﻟﻜﯿﻤﯿﺎء- ﻛﻠﯿﺔ اﻟﻌﻠﻮم‬ ‫ﺟﺎﻣﻌﺔ اﻟﻘﺎھﺮة‬ ‫6002‬