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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 اهللا الرمحن الرحیم
رب اشرح ىل صدرى"ویسرىل أمرى
واحلل عقدة من لساىن یفقهوا
"قوىل صدق اهللا 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.
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, Abo-
Naglla A. 2005. Application of a circulating antigen detection immunoassay
for laboratory diagnosis of extra-pulmonary and pulmonary tuberculosis.
Clin Chim Acta, 356: 58-66.
Note
i
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
L J 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
ii
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-
PAGE
Sodium dodecyl sulfate- polyacrylamide gel
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
iii
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
2. Tuberculosis 7
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
3. Diagnosis of tuberculosis 21
3.1. Microscopic method 21
iv
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 34
3.8.1. Application of monoclonal antibodies in diagnosis of M. tuberculosis antigen
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
v
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
vi
List of figures
Fig. no. Title page
1 Epidemiology of TB in the world 9
2 Mycobacterium tuberculosis stained with Ziehl-
Neelsen staining 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
6
Coomassie blue stained SDS-PAGE of sera from
pulmonary tuberculosis patients and non infected
individuals under reducing conditions
65
7
Immunoblots of TB-55 mAb target antigen in sera of
pulmonary tuberculosis patients and non infected
individuals 66
8
Coomassie blue stained SDS-PAGE of sera from
extra-pulmonary tuberculosis patients and non
infected individuals under reducing conditions
68
9
Immunoblots of TB-55 mAb target antigen in sera of
extra pulmonary tuberculosis patients and non
infected individuals
69
10
Coomassie blue stained SDS-PAGE of CSF from
tuberculous meningitis patients and non-tuberculous
CSF under reducing conditions
71
11
Immunoblots of TB-55 mAb target antigen CSF from
tuberculous meningitis patients and non-tuberculous
CSF 72
vii
Fig. no. Title page
12
Coomassie blue stained SDS-PAGE of tuberculous
ascetic fluid from peritonitis tuberculosis patients and
non-tuberculous ascites fluid under reducing
conditions
74
13
Immunoblots of TB-55 mAb target antigen in
tuberculous ascetic fluid from peritonitis tuberculosis
patients and non-tuberculous ascites fluids
75
14 Coomassie stain SDS-PAGE of purified 55 kDa
antigen from pulmonary tuberculosis sera 77
15 Coomassie stain SDS-PAGE of purified 55 kDa
antigen from extra-pulmonary tuberculosis sera 78
16 Coomassie stained SDS-PAGE of purified 55 kDa
antigen from CSF 79
17 Coomassie stain SDS-PAGE of purified 55 kDa
antigen from ascites. 80
18 Capillary electrophoresis (CE) electropherogram of
purified 55 kDa antigen 81-84
19 Reactivity of the purified 55 kDa antigen against TB-
55 monclonal antibody using dot-ELISA 86
20 The relative percentages of the amino acid
concentrations of the purified 55 kDa antigen 91
viii
Fig. no. Title page
21 Types of tuberculosis 93
22 Dot-ELISA of serum samples from tuberculosis
patients and non-infected individuals 96
23
Levels of circulating 55-kDa antigen detection using
Dot- ELISA in serum samples of pulmonary
tuberculosis patients
98
24
Levels of circulating 55-kDa antigen detection using
Dot- ELISA in serum samples of extra-pulmonary
tuberculosis patients
103
25
Overall levels of circulating 55-kDa antigen detection
using Dot- ELISA in serum samples of pulmonary
and extra-pulmonary tuberculosis patients
106
26 Advantages of circulating 55-kDa antigen detection
by using Dot- ELISA in 506 serum samples 108
ix
List of tables
Table no. Title page
1 Some available antibody tests for diagnosis of
pulmonary Tuberculosis 32
2 Rf values of unknown antigens and of standards protein
mixture 62
3 Partial biochemical nature of the purified 55-kDa
antigens reactive epitope 88
4 Amino acid concentrations of the purified 55 kDa
antigen 90
5 The types of extra-pulmonary tuberculosis according to
sites of infection in 93 serum samples 94
6
Advantages of circulating 55-kDa antigen detection by
using Dot-ELISA in serum samples of pulmonary
tuberculosis
98
7 Detailed analysis of extra-pulmonary tuberculosis
using dot ELISA 102
8
Advantages of circulating 55-kDa antigen detection by
using Dot-ELISA in serum samples of extra-pulmonary
tuberculosis
104
9 Advantages of circulating 55-kDa antigen detection by
using Dot- ELISA in serum samples 107
Introduction and Aim of work
1
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
Introduction and Aim of work
2
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.
Review of literature
3
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
Review of literature
4
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
Review of literature
5
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 anti-
tuberculous activity of weak acids correlated with their ability to disrupt the
membrane potential but not the internal pH (Zhang et al., 2003).
Review of literature
6
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).
Review of literature
7
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,
Review of literature
8
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 smear-
positive 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 socio-
economic 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
Review of literature
9
Figure 1. Epidemiology of TB in the world (WHO, 2000).
Review of literature
10
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
Review of literature
11
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
Review of literature
12
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).
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13
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
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14
(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
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15
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
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16
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 soft-
tissue 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
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17
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
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18
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).
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19
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 Calmette-
Guerin) vaccine was developed from an attenuated strain of M. bovis at the
beginning of the twentieth century. Its widespread use as a vaccine against
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20
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).
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21
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 acid-
fast 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 acid-
fast bacteria in tissue specimens is critical (Bishop and Neumann, 1970). In
1882, Ehrlich discovered that Mycobacterium with fuchsin (in the presence of
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22
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 carbol-
fuchsin 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 NaOCl-
treated 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,
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23
Figure 2. Acid fast bacilli (shown in red) are tubercle bacilli (Grange, 2002)
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24
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 against the dark
background (Chessbrough, 2000 and Murray et al., 2003).
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25
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
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26
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
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27
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 ; Somoskvِi and Magyar,
1999; Hanna et al., 1999 and Heifets et al., 2000). Though these methods
offer an attractive enhancement (not replacement) to culture on Lwِenstein-
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
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28
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
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29
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
Review of literature
30
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
Review of literature
31
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. Cross-
reaction 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.
Review of literature
32
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)
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33
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
Review of literature
34
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 drug-
susceptibility 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
Review of literature
35
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
Review of literature
36
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
Review of literature
37
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.
Materials and Methods
38
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 extra-
pulmonary 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
Materials and Methods
39
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
Materials and Methods
40
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:
Materials and Methods
41
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
Materials and Methods
42
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 (SDS-
PAGE):
BCG, ascites fluid, CSF and serum samples at 30 µg/lane were separated
by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
Materials and Methods
43
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),
Materials and Methods
44
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.
Materials and Methods
45
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
Materials and Methods
46
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
Materials and Methods
47
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
Materials and Methods
48
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.
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.
Migrated distance of protein
Migrated distance of dye Rf =
Materials and Methods
49
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
Materials and Methods
50
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 computer-
controlled 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
Materials and Methods
51
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
Materials and Methods
52
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
Materials and Methods
53
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
Materials and Methods
54
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
Materials and Methods
55
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
Materials and Methods
56
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
Materials and Methods
57
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:
Evaluated test Reference
Test + ve - ve
Total
+ 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.
Materials and Methods
58
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.
Results
59
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.
Results
60
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).
Results
61
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.
Results
62
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
Unknown antigen
(82 kDa) 1.91 0.14
Unknown antigen
(55 kDa) 1.74 0.31
Results
63
Figure 5. Liner calibration of standard molecular weights.
Phosphorylase B
Ovalbumin Glutamate dehydrogenase
Aldolase
Bovine serum albumin
Soybean trypsin inhibitor
Carbonic anhydrase
Results
64
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.
Results
65
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).
Results
66
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).
Results
67
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.
Results
68
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).
Results
69
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).
Results
70
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 non-
tuberculous neurological diseases patients from control individuals were
observed as shown in figure 11.
Results
71
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).
Results
72
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).
Results
73
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) from control
individuals were observed as shown in figure 13.
Results
74
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).
Results
75
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 % 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. 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).
Results
76
Part 2
Purification and characterization of the circulating 55 kDa
antigen from different body fluids of pulmonary and extra-
pulmonary 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.
Results
77
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).
Results
78
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).
Results
79
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).
Results
80
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).
Results
81
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 75-
µm capillary, 30 kV, 20 oC and UV detection at 200 nm.
A
Results
82
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.
B. Purified antigen from extra-pulmonary tuberculosis
B
Results
83
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.
C
Results
84
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.
D
Results
85
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.
Results
86
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).
Results
87
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.
Results
88
Table 3. Partial biochemical characterization of the reactive epitope on
purified 55-kDa antigen isolated from different sources (serum,
CSF, ascites) .
Treatments
Reactivity of
purified antigen
using dot-ELISA
Type of reagents Concentrations Incubation
time Treated Untreated
Acid 0.2 M HCl 1 hour -Ve + Ve
Base 0.2 M NaOH 1 hour -Ve + Ve
Trichloroacetic acid 40% 15 min.
a- precipitate + Ve -
b- Supernatant -Ve -
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
Results
89
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 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% as shown in table 4 and figure 20. So, the 55- kDa antigen is a basic
polypeptide chain with a hydrophilic nature.
Results
90
Table 4. Amino acid concentrations of the purified 55 kDa M. tuberculosis
antigen.
Type
Name
Concentration
(nmol/mg protein) %
Hydrophobic
Leucine
Isoleucine
Valine
Proline
Methionine
Tyrosine
Alanine
47.14
49.28
81.42
59.28
98
94.28
113.14
24.6 %
Hydrophilic
Glycine
Serine
Therionine
755.71
214.28
55.71
46.4 %
Basic
Lysine
Arginine
Histidine
162.85
85.71
111.42
16.3 %
Acidic
Glutamic acid
Aspartic acid
135.71
142.85
12.7 %
Results
91
05
101520253035404550
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.
Hydrophobic Hydrophilic Basic Acidic
Perc
enta
ge (%
)
24.6 %
46.4 %
16.3 % 12.7 %
Types of amino acides
Results
92
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.
Results
93
76 %
24 %
0102030405060708090
100
Figure 21. Types of tuberculosis. Of the 389 tuberculosis cases examined,
296 were pulmonary tuberculosis (76 %) and 93 cases (24 %) were extra-
pulmonary tuberculosis.
Pulmonary TB Extra-Pulmonary TB
Perc
enta
ge (%
)
Types of tuberculosis
Results
94
Table 5. The types of extra-pulmonary tuberculosis according to sites of
infection in 93 serum samples.
% No. Types of extra-pulmonary tuberculosis
27 25 TB peritonitis
24 22 TB meningitis
20 19 TB of genitourinary tract
16 14 TB lymphadenitis
5 5 Pott's disease
3 3 TB sinusitis
3 3 TB arthritis
2 2 Milliary TB
Results
95
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.
Results
96
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+).
Results
97
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.
Results
98
13%
73%
14%
0
10
20
30
40
50
60
70
80
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 (+++/++++)
Negative Low Antigen level* High Antigen level**
Perc
enta
ge %
Results
99
Table 6. Advantages of circulating 55-kDa antigen detection by using Dot-
ELISA in serum samples of pulmonary tuberculosis.
TB-Ag detection Clinical diagnosis (gold standard)
+ - Total
Pulmonary tuberculosis patients 257 (a) 39 (c) 296
Patients with respiratory diseases other
than TB and Healthy controls 4 (b ) 113 (d ) 117
Total 261 152 413
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 %
Results
100
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 55-
kDa (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 55-
kDa (89%), 12 out of 14 of TB lymphadenitis were positive for circulating 55-
kDa (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.
Results
101
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.
Results
102
Table 7. Detailed analysis of extra-pulmonary tuberculosis using dot
ELISA.
% +ve No. +ve
samples
No. of
serum samples
Types of extra-pulmonary
tuberculosis
88 22 25 TB peritonitis
91 20 22 TB meningitis
89 17 19 TB genitourinary tract
86 12 14 TB lymphadenitis
100 5 5 pot's disease
100 3 3 TB sinusitis
100 3 3 TB arthritis
100 2 2 Milliary TB
Results
103
10%
62 %
28%
0
10
20
30
40
50
60
70
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 (+++/++++)
Negative Low Antigen level* High Antigen level**
Perc
enta
ge %
Results
104
Table 8. Advantages of circulating 55-kDa antigen detection by using Dot-
ELISA in serum samples of extra-pulmonary tuberculosis.
TB-Ag detection Clinical diagnosis (gold standard)
+ - Total
Extra-pulmonary tuberculosis patients 84 (a) 9 (c) 93
Patients with respiratory diseases other than
TB and Healthy controls 4 (b ) 113 (d ) 117
Total 88 122 210
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 %
Results
105
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.
Results
106
12.3 %
70.2 %
17.5 %
0
10
20
30
40
50
60
70
80
Figure 25. Overall levels of circulating 55-kDa antigen detection using Dot-
ELISA in serum samples of pulmonary and extra-pulmonary tuberculosis
patients.
** Weak positive test (+/++)
*** Strong positive test (+++/++++)
Negative Low Antigen level* High Antigen level**
Perc
enta
ge %
Results
107
Table 9. Overall Advantages of circulating 55-kDa antigen detection by
using Dot- ELISA in serum samples.
TB-Ag detection Clinical diagnosis (gold standard)
+ -
Total
TB patients 341 (a) 48 (c) 389
Patients with respiratory diseases other than
TB and Healthy controls 4 (b ) 113 (d ) 117
Total 345 161 506
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%
Results
108
0
10
20
30
40
50
60
70
80
90
100
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
Sensitivity Specificity Efficiency PPV* NPV**
88 % 97 %
90 % 99 %
70 %
Perc
enta
ge %
Discussion
109
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.,
Discussion
110
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.
Discussion
111
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.
Discussion
112
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
Discussion
113
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
Discussion
114
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 CSF-
cytospin 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 TB-
55 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
Discussion
115
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
Discussion
116
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 reverse-
phase 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
Discussion
117
to be a fibronectin-binding protein as determined by ELISA and could be
purified by affinity chromatography with fibronectin-Sepharose. A similar 66-
kDa 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 interferon-
gamma 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
Discussion
118
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,
Discussion
119
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 TAS-
1D3, 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, sulfur-
containing 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
Discussion
120
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%).
Discussion
121
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
Discussion
122
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
Discussion
123
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
Discussion
124
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 smear-
negative 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 anti-
BCG immunoglobulin. BCG protein standard was used as positive control.
Thirty-nine patients with culture-proven pulmonary tuberculosis were tested.
Discussion
125
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
Discussion
126
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.
Discussion
127
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 86-
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 (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 extra-
pulmonary 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,
Discussion
128
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 dot-
immunobinding 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).
Discussion
129
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 55-
kDa 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
Discussion
130
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.
Summary
131
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.
Summary
132
2- The 55–kDa target antigen was purified from ascites fluid and CSF of extra-
pulmonary tuberculosis and serum samples of pulmonary and extra-
pulmonary 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
Summary
133
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 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. 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
Summary
134
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 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 extra-
pulmonary 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
Summary
135
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.
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136
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الملخص العربي
1
الملخص العربي
من أخطر االمراض البكتیریة التى تصیب حوالى ثلث سكان العالم ویتسبب )السل (الدرن
ملی ون ش خص حی ث ت صیب بكتیری ا ال درن الرئ ھ ف ى 3ص ابة جدی دة ووف اة إ ملی ون 8 ف ى سنویا
تم د ت شخیص ویع. ف ى ب اقى الح االت من الحاالت أو تنتقل الى أعضاء الجسم االخ رى % 80-84
عل ى الفح ص ال سریرى والفحوص ات التشخ صیة (pulmonaly tuberculosis)ال درن الرئ وى
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
، Polymerase chain reactionالحامض النووى لبكتیریا الدرن باستخدم تفاعل البلم ره المتسل سل
م ن ال درن ًا فان ھ أكث ر تعقی د (Extra-pulmonaly tuberculosis)شخیص ال درن خ ارج الرئ ة أما ت
وح دیثا . م فحصھا باثولوجی ا ث (Biopsy) العضو المصاب نسیجمن الرئوى النھ یعتمد على عینة
تعتم د عل ى أس تخدام ج سم م ضاد وھ ى األلی زا النقطی ة ت م تط ویر طریق ة مناعی ة ب سیطة وس ھلة
عین ات س یرم للمرض ى ف ي ) كیلودالت ون 55 (ال درن ات بكتیریا أنتیجینأحد سیلة لتعیین أحادى الن
.الرئويمصابین بالدرن
الج سم س وائلف ي كیلودالت ون 55 أنتیج ین ال درنالتع رف عل ى إل ىوتھ دف ھ ذه الدراس ة
مرض ى م صابین النتج ین ف ى عین ات س یرم ل اوتقی یم كفائ ة أختب ار األلی زا النقطی ة ف ى تعی ین ھ ذا
. الرئوى وخارج الرئةبالدرن
:اآلتيوقد اشتملت ھذه الدراسة على
بال درن س یرم المرض ى الم صابین بال درن الرئ وي و عینات و) BCG(لقاح جینات یانتفصل تم . 1
باس تخدام طریق ة ) ascites( وس ائل االست سقاء )CSF(خ ارج الرئ ة و س ائل النخ اع ال شوكى
ث م التع رف ) (polyacrylamide gel electrophoresis تروف وریزسالب ولي أكریلمی د ج ل الك
ث م التع رف Coomassie blueعلى البروتینات المفصولة بصبغھا ب صبغة الكوماس ى الزرق اء
الخاص بالج سم الم ضاد أح ادى Reactive epitope على األنتیجین الذي یحوى الجزء الفعال
س تخدام طریق ة ال شفط المن اعي با تل ك العین ات ف ي TB- 55 mAbالن سیلة
الملخص العربي
2
Immunoblotting technique ثم تعی ین ال وزن الجزئ ي النتیج ین ال ذي یح وى الج زء الفع ال
وج د وقد الخاص بالجسم المضاد باستخدام خلیط من بروتینات قیاسیة معلومة الوزن الجزئي
أنتج ین إلض افة إل ى باف ي تل ك العین ات كیلودالت ون 55ي لھذا األنتیجین ھ و یأن الوزن الجزئ
).(BCGكیلودالتون في لقاح 82أخر ذو وزن جزئیى
وم ن س یرم الرئ وي الم صابین بال درن سیرم المرض ى كیلودالتون من 55 األنتیجین تنقیة تم -2
االست سقاء ال شوكى وس ائلم ن س ائل النخ اع خ ارج الرئ ة و الم صابین بال درنالمرض ى
الجی ل م ن تنقیت ة ث م )(Preparative gel gelelectrophoresisالتح ضیري ج لال باس تخدام
الطبیع ة ھ ذه األنتیجین ات لمعرف ةتوص یف ت م ) Electroelution( اإلزاح ة الكھربی ة بطریق ة
الج سم الم ضاد أح ادى الن سیلة باس تخدام Reactive epitope الكیمیائی ة للج زء الفع ال
TB- 55 mAb البیتی دات وتعط ى ة ع ن سل سلة مف رده عدی دة وجد أن ھذه األنتیجینات عب ار ف
جھ از عل ى عن د الف صل عن د اا دقیق ةواح دة )peak(قم ة
)Capillary zone electrophoresis(یترس ب بم ادة و Tricholoroacetic acid یت أثر ال
حللة للبروتین ات ووج د أی ضا أن فعالی ة ویتحلل باألنزیمات المPeriodate باألكسدة باستخدام
وال یت أثر )(Electrophoretic migration ھ ذه األنتیجین ات ال تت أثر ب الھجرة الكھربی ة
مم ا ی دل عل ى ) Mercaptoethanolأو Sodium dodecyl sulfate( بالعوام ل المختزل ة مث ل
لعین ة حم اض األمینی ة المكون ة تم عمل تحلیل األ. للجزء الفعال ھذه األنتیجینات العاليالثبات
الكروم اتوجرافى ال سائل ع الى الكف اءة جھ از التحلی لاألنتیجین ات باس تخدام م ن
High performance liquid chromatograph أمین ى ن سبھ حم ض 15وج د أن ھ یتك ون م ن ف
ب ة للم اء ھ ى ون سبھ األحم اض األمینی ة المح % 24.6األحم اض األمینی ة الكارھ ة للم اء ب ھ
ونسبة األحماض األمینی ة % 16.3ونسبة األحماض األمینیة ذات الخواص القاعدیة % 46.4
%.12.7ذات الخواص الحمضیة
الملخص العربي
3
ال دوار ج ین ال درن ی للك شف ع ن انت TB- 55 mAb تم استخدام الجسم المضاد أحادى الن سیلة -3
وھ و اختب ار )dot-ELISA( اإللی زا النقطی ة بطریق ة ف ي ال سیرم كیلودالت ون 55
)Semi-quantitative ( حیث یظھر ل ون بنف سجي ف ي عین ة ال شخص الم صاب ببكتیری ا ال درن
غیر مصاب وتختلف درج ة الل ون ف ي العین ة الموجب ة م ن الوال یظھر لون في عینة الشخص
ین ال درنج یانتح سب م ستوى ) 4+،+3( إل ى الدرج ة القوی ة ) 2+،+1(الدرج ة ال ضعیفة
حال ة م صابة 389 حال ة م نھم 506 ت م أس تخدام حی ث ف ي ال سیرم كیلودالت ون55 ال دوار
حال ة م ن مرض ى ام راض تنف سیة ص دریة غی ر ال درن وأص حاء كمجموع ة 117بال درن و
حال ة م صابة بال درن الرئ وى 296م صابة بال درن م نھم لح االت س یرم ھ عین 389ض ابطة،
ین بال درن خ ارج الرئ ھ ال ذین ت م تق سیمھم ح سب موض ع م صاب %) 24( حالة 93، %) 76(
حال ھ م صابة بال درن ال سحائى بن سبة 22، %) 27( حالة درن بطن ى بن سبة 25االصابة الى
حال ھ 19، %) 20(بن سبة حال ة م صابة بال درن ف ى القن اة البولی ة التناس لیة 19، %) 24(
بن سبة ح االت م صابة بم رض ب وتس 5، %) 16( بن سبة الیمفاوی ة مصابة بالدرن ف ى الغ دد
ح االت م صابة ب درن ف ى 3 و )%3( بن سة ب درن ف ى المفاص ل ح االت م صابة3 ، )5%(
بن سبة ض ع مختلف ة م ن الج سم ا ب درن ف ى مو ین م صابت ین وح الت )%3( بنسبة الجیوب األنفیة
)2(% .
296 حال ة م ن 257یوج د ف ى كیلودالت ون 55 ال دوار الدرن انتیجینفاوضحت النتائج أن
حال ة م صابة بال درن الرئ وى ال یوج د 39و %) 87(حال ھ م صابة بال درن الرئ وى بن سبة
ولتقییم كفائة أختبار األلیزا النقطیة فى . %)13(بنسبة كیلودالتون 55 الدوار الدرن انتیجین
م صابین ن ف ى عین ات س یرم لمرض ى م صابی كیلودالت ون 55 ال درن ال دوارانتیج ینتعی ین
حال ة 296أنتجین الدرن الدوار من حالة یوجد بھا 257 الرئوى فأوضحت النتائج أن بالدرن
حالة من المجموعة ال ضابطة 117 حالة من 113، %) 87حساسیة ( الرئوى مصابھ بالدرن
ال یوج د بھ ا أنتج ین ال درن ) غیر الدرن وأشخاص طبیعین صدریةحاالت مصابة بأمراض (
الملخص العربي
4
% . 74وقیم ة تنبؤی ة س البة % 98وقیم ة تنبؤی ة موجب ة %90وكفائة %) 97خصوصیة (
39: حال ة م صابة بال درن الرئ وى ك االتى 296 ال درن فكان ت نت ائج انتیجینتم تحدید مستوى
حالة یوجد بھا م ستوى ض عیف 215و%) 13( الدرن الدوار بنسبة انتیجینحالة الیوجد بھا
نتیجین حالة یوجد بھا مستوى قوى ال42األضافة الى ب%) 73( الدرن الدوار بنسبة نتیجینال
%)14(بنسبة الدرن الدوار
ف ى انتیج ینكطریق ة س ھلة وب سیطة للك شف ع ن )(dot ELISAت م اس تخدام االلی زا النقطی ة . 4
ال دوار ال درن انتیج ین عینات سیرم حاالت مصابة بالدرن خارج الرئ ة فاوض حت النت ائج أن
%) 90( حالھ مصابة بالدرن خ ارج الرئ ة بن سبة 93 حالة من 84 فى یوجد كیلودالتون 55
بن سبة كیلودالت ون 55 ال دوار ال درن انتیجین حاالت مصابة بالدرن خارج الرئة ال یوجد 9و
كیلودالت ون 55 ال درن ال دوار انتیج ین ولتقییم كفائة أختبار األلیزا النقطیة فى تعیین %). 10(
حال ة یوج د 84 خ ارج الرئ ة فأوض حت النت ائج أن بالدرنن فى عینات سیرم لمرضى مصابی
حال ة 113، %) 90ح ساسیة ( الرئ وى مصابھ بال درن حالة 93أنتجین الدرن الدوار من بھا
غی ر ال درن ص دریة ح االت م صابة ب أمراض ( حال ة م ن المجموع ة ال ضابطة 117م ن
وقیم ة % 94ة وكفائ %) 97خ صوصیة (ال یوج د بھ ا أنتج ین ال درن ) وأش خاص طبیع ین
ال درن فكان ت انتیج ینت م تحدی د م ستوى % . 93وقیم ة تنبؤی ة س البة % 95تنبؤی ة موجب ة
ال درنانتیج ین ح االت ال یوج د بھ ا 9: حال ة م صابة بال درن خ ارج الرئ ة ك االتى 93نت ائج
بن سبة ال دوار ال درننتیج ینال حال ة یوج د بھ ا م ستوى ض عیف 58و%) 10( بن سبة ال دوار
بن سبة ال دوار ال درننتیج ینال حال ة یوج د بھ ا م ستوى ق وى 26ألض افة ال ى با%) 62(
)28.(%
الرئ وى وخ ارج الرئ ة م صابین بال درنلمرض ى نتیج ین ال درنأل الكل ىم ستوىال ت م تحدی د .5
ال دوار ال یوج د بھ ا انتیج ین ال درن ل ة حا 48: حال ة م صابة بال درن ك االتى 389فكانت نتائج
بن سبة ال دوار ال درن النتیج ین ل ة یوج د بھ ا م ستوى ض عیف حا273و%) 12.3(بن سبة
الملخص العربي
5
بن سبة ال دوار ال درنالنتیج ین حال ة یوج د بھ ا م ستوى ق وى 68باألض افة ال ى %) 70.2(
55 الدرن الدوار انتیجینكفائة أختبار األلیزا النقطیة الكلیة فى تعیین ولتقییم %).17.5(
الرئ وى وخ ارج الرئ ة فأوض حت بال درنن عین ات س یرم لمرض ى م صابی ف ىكیلودالت ون
م صابة بال درنل ة حا389 حال ة م ن 341یوج د ف ى ال دوار ال درنانتیج ینأن النت ائج
مرض ى ام راض ( حال ة م ن العین ات ال ضابطة 117م ن فق ط ح االت 4و %) 88 حساسیة(
وكفائ ة%) 97خ صوصیة ( ال دوار ال درنانتیج ینیوج د بھ ا ) تنف سیة غی ر ال درن وأص حاء
ال دوار وكانت القیمة التنبؤیة الموجب ة النتیج ین ال درن %) 90( الدوار الدرن انتیجین أختبار
. %73 الدوار و القیمة التنبؤیة السالبة النتیجین الدرن % 99
:والخالصة
ال سیرم عین ات ف ى كیلودالت ون 55 ال دوارنتیج ین ال درنوتوص یف أ و تنقی ة ت م تعری ف
الم صابین بال درن وبالت الى یمك ن اس تخدامھ ىلمرض لى وس ائل األست سقاء وس ائل النخ اع ال شوك
ال دوار ال درن انتیج ین ع ن للك شف طریقة اإللیزا النقطیة .ككاشف فى التشخیص المناعى للدرن
طریق ة ذات درج ات عالی ة م ن الرئ وى وخ ارج الرئ ة بال درن فى عینات سیرم لمرضى مصابین
ویمكنھ ا ت شخیص أن واع مختلف ة م ن ال درن خ ارج الرئ ة % 95 والخ صوصیة % 88 الحساسیة
م رض ودرن الغ دد الیمفاوی ة ودرن القناة البولیة التناس لیة و الدرن السحائى و بطنىالدرن مثل ال
باألض افة ال ى ضع مختلفة م ن الج سم ادرن فى موو درن الجیوب األنفیة و درن المفاصل و بوتس
وطبق ا لتوص یات منظم ة ال صحة العالمی ة . جھ زة معق دة غیر مكلفة وال تحتاج إل ى أ وأنھا سریعة
) زراع ة بكتیری ا ال درن ( یمك ن أن ی ستبدل الطریق ة القیاس یة لت شخیص ال درن فان األختبار ال ذى
طریق ة اإللی زا ین صح باس تخدام ول ذلك % 95 وخ صوصیتة % 80البد أن تكون درج ة حاس یتة
.فى الكشف المبكر عن مرضى الدرن النقطیة
المستخلص محمد مصطفى عمران : االسم
دراسات كیمیائیة حیویة على أحد أنتیجینات بكتیریا التدرن الرئوي : عنوان الرسالة
)كیمیاء حیویة( دكتورالفلسفة فى العلوم :الدرجة
وف ى ھ ذه التع رف عل ى أنتیج ین ال درن خط وه ھام ة نح و تشخی صھ ال دقیق : ملخ ص البح ث
أنتیج ین ال درن ف ي عین ات ال سیرم وس ائل االست سقاء وس ائل النخ اع عل ى تع رفالت م الدراس ة
وتوص یفھ جزئی ا ث م تنقیت ھ طریق ة ال شفط المن اعي والشوكى باستخدام جسم مضاد أح ادى الن سیلة
طریقة مناعیة س ھلة وب سیطة تم استخدام الیزا النقطیة ك. كیلو دالتون55كبروتین لھ وزن جزیئي
م ن ح االت ال درن خ ارج % 90 حی ث ثب ت وج وده ف ى عین ات ال سیرم تعیین أنتیجین الدرن في ل
عالی ة من حاالت ال درن الرئ وي وق د أظھ رت ھ ذه الطریق ة درج ة خ صوصیة % 87الرئة وفى
و تعیینھ نتیجین الدرنتم تعریف وتوصیف جزئیي أل : الخالصة. في المجموعة الضابطة % 97
ف ي الت شخیص العالی ة ةات الح ساسیة والخ صوصیف ي ال سیرم باس تخدام طریق ة الی زا النقطی ة ذ
. للدرنالروتیني ولتدعیم التشخیص االكلینكى
، سیرم كیلو دالتون55الدرن ، التشخیص، أنتیجین ، :الكلمات الدالھ
توقیع السادة المشرفون
............................................................... د سناء عثمان عبد اهللا.ا - 1
............................................................... د عبد الفتاح محمد عطا اهللا.ا - 2
............................................................... عمرو سعد محمد.د - 3
یعتمد ،،،،
لرفعت حسن ھال/ د .أ
رئیس مجلس قسم الكیمیاء
جامعة القاھرة – العلوم كلیة
حیویة على أحد أنتیجینات ةدراسات كیمیائی
بكتیریا التدرن الرئوي
رسالة مقدمة
للحصول على درجة دكتور فلسفة العلوم في الكيمياء الحيوية
من الطالب
محمد مصطفى عمران دمیاط الجدیدة– مركز أبحاث التكنولوجیا الحیویة
كلیة العلوم -الكیمیاءقسم
جامعة القاھرة
2006