Diagnosis of Mycobacterium Tuberculosis

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


antigen from extra-pulmonary tuberculosis sera 78

16 Coomassie stained SDS-PAGE of purified 55 kDa

antigen from CSF 79


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


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


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


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


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).


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).


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,


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


9

Figure 1. Epidemiology of TB in the world (WHO, 2000).


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


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


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).


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


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


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


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


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


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).


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


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).


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


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,


23

Figure 2. Acid fast bacilli (shown in red) are tubercle bacilli (Grange, 2002)


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).


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


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


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


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


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


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


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 immunoassay

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.


32

Table 1. Some antibody tests for diagnosis of pulmonary tuberculosis


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)


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


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


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


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


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


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


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


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


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:


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


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)


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),


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.


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


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


1- Tris buffered saline (TBS), pH 7.4:

12.11 gm trisma base + 11.688 gm Sodium chloride were dissolved in


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


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 =


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


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


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


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



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:


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:


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:



same volume of 40 % TCA. After incubation at room temperature for 30


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:


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:


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


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


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.


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


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.


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.


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


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.


nitrocellulose (NC) paper. TB–55 mAb was used as a primary antibody, and


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


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),


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.)



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.)



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


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


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


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),


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


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 (+++/++++)


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.


+ - 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 %,


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.


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 (+++/++++)


Perc

enta

ge %

Results

107

Table 9. Overall Advantages of circulating 55-kDa antigen detection by

using Dot- ELISA in serum samples.


+ -

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 %,


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


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.

References

136

References

Aggarwal P, Wali JP, Singh S, Handa R, Wig N and Biswas A. 2001. A

clinico-bacteriological study of peripheral tuberculous lymphadenitis. J

Assoc Physicians India, 49: 808-812.

Aguilar LD, Infante E, Bianco MV, Cataldi A, Bigi F and Pando RH. 2006.

Immunogenicity and protection induced by Mycobacterium tuberculosis

mce-2 and mce-3 mutants in a Balb/c mouse model of progressive

pulmonary tuberculosis. Vaccine, 20: 2333-2342.

Aitken, A and Learmonth M. 1996. Quantition of tryptophan in proteins. In:

Walker M. Introduction of protein protocol hand book. PP: 29-32. Humana

press. USA.

Al-Hakeem MM, Chaudhary AR, Aziz S and Al-Aska AK. 2005. Frequency

of isolated positive sputum cultures among pulmonary tuberculosis

patients. Saudi Med J, 26: 634-640.

Al-Jahdali H, Memish ZA and Menzies D. 2003. Tuberculosis in association

with travel. Int J Antimicrob Agents, 21: 125-130.

Al-Orainey IO, Gad el Rab MO, al-Hajjaj MS and Saeed ES. 1992.

Detection of mycobacterial antigens in sputum by an enzyme

immunoassay. Eur J Clin Microbiol Infect Dis,11: 58-61.

Altet-Gomez MN, Alcaide J, Godoy P, Romero MA and Hernandez del Rey

I. 2005. Clinical and epidemiological aspects of smoking and tuberculosis:

a study of 13,038 cases. Int J Tuberc Lung Dis, 9: 430-436.

Anand PK, Kaul D and Sharma M. 2006. Green tea polyphenol inhibits

Mycobacterium tuberculosis survival within human macrophages. Int J

Biochem Cell Biol, 38: 600-609.

References

137

Anane R. 2003. Childhood tuberculosis in Africa: epidemiologic, clinical and

therapeutic aspects. Med Trop, 63: 473-480.

Anderson P, Munk ME, Pollock JM and Doharty TM. 2000. Specific

immune-based diagnosis of tuberculosis. Lancet, 356: 1099-10104.

Andrews AT. 1986. Electrophoresis: Theory techniques and biochemical and

clinical application. PP: 213-242. 2nd ed. Oxford University presses. New

York.

Arais LM, Nguyen LN Ho, Kuijper S, Jensen HM and Kolk AH. 2000.

Development of antigen detection assay for diagnosis of tuberculosis using

sputum samples. J Clin Microbiol, 38: 2278-2283.

Ashok M, Neelam K and Beena KR. 2002. Immunohistochemical detection of

Mycobacterial antigen in tuberculous lymphadenitis. Ind J Tub, 49: 213-

216.

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.

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.

Avdienko VG, Kondrashov SI and Liashenko SM. 1996. An analysis of the

specificity of anti-Mycobacterium monoclonal antibodies and of the

chemical nature of the antigenic determinants with which they react. Probl

Tuberk, 33: 6-8.

Balian A, de Pinieux I, Belloula D, Barthelemy P, Montembault S, Girard

T, Raynard B, Le Gall C, Capron F, Naveau S and Chaput JC. 2000.

References

138

Abdominal tuberculosis: deceptive and still encountered. Presse Med, 29:

994-996.

Banchuin N, Wongwajana S, Pumprueg U and Jearanaisilavong J. 1990

Value of an ELISA for mycobacterial antigen detection as a routine

diagnostic test of pulmonary tuberculosis. Asian Pac J Allergy Immunol, 8:

5-11.

Banerjee S, Kumar S and Harinath BC. 2003. Isolation and characterization

of in vivo released 41 kDa Mycobacterial antigen in pulmonary and bone

and joint tuberculosis and its identification with H37Ra in vitro released

antigen. Int J Tuberc Lung Dis, 7: 278-283.

Barbolini G, Bisetti A, Colizzi V, Damiani G, Migaldi M and Vismara D.

1989. Immunohistologic analysis of mycobacterial antigens by monoclonal

antibodies in tuberculosis and mycobacteriosis. Hum Pathol, 20: 1078-

1083.

Bathamley GH, 1995. Serological diagnosis of tuberculosis. Eur Resp J, 8:

676.

Baylan O. 2005. Culture based diagnostic methods for tuberculosis. Microbiol

Bull, 39: 107-124.

Beck ST, Leite OM and Arruda RS. 2005. Combined use of western

Blot/ELISA to improve the serological diagnosis of human tuberculosis.

BJID, 9: 35-43.

Beltran S, Douadi Y, Lescure FX, Hanau M, Laurans G, Ducroix JP. 2003.

A case of tuberculous sinusitis without concomitant pulmonary disease. Eur

J Clin Microbiol Infect Dis, 22 :49-50.

References

139

Bengis R. 2000. Tuberculosis in free-ranging mammals. In: Fowler M and

Miller RE (edts). Zoo and wildlife medicine: current therapy. pp:101–113.

W.B. Saunders Company; Philadelphia.

Bhargava DK, Dasarathy S, Shriniwas MD, Kushwaha AKS, Duphare H,

Kapoor BML. 1992. Evaluation of enzyme-linked immunosorbent assay

using mycobacterial salineextracted antigen for the serodiagnosis of

abdominal tuberculosis. Am J Gastroenterol, 87 : 105-108.

Bhaskar S, Khanna SP and Mukherjee R. 2000. Isolation, purification and

immunological characterization of novel low molecular weight protein

antigen CFP 6 from culture filtrate of M. tuberculosis. Vaccine, 18: 2856-

2866.

Bishop BJ, and Neumann G. 1970. The history of Ziehl - Neelsen stain.

Tubercle, 51: 196 - 206.

Blanie M, Pellegrin JL and Maugein J. 2005. Contribution of PCR in the

diagnosis of extra-pulmonary tuberculosis. Med Mal Infect,35 :17-22.

Bothamley GH. Serological diagnosis of tuberculosis. 1995. Eur Respir J

Suppl, 20: 676-688.

Brassard P, Steensma C, Cadieux L and Lands LC. 2006. Evaluation of a

school-based tuberculosis-screening program and associate investigation

targeting recently immigrated children in a low-burden country. Pediatrics,

117:148-156.

Broccolo F, Scarpellini P, Locatelli G, Zingale A, Brambilla AM, Cichero P,

Sechi LA, Lazzarin A, Lusso P and Malnati MS. 2003. Rapid diagnosis

of Mycobacterial infections and quantitation of Mycobacterium

References

140

tuberculosis load by two real-time calibrated PCR assays. J Clin Microbiol,

41: 4565-4572.

Brock I, Munk ME, Kok-Jensenand A and Anderson P. 2001. Performance

of whole blood IFN-gamma test for tuberculosis diagnosis based on PPD

or the specific antigens ESAT-6 and CFP-10. Int J Tuberc Lung Dis, 5:

462-467.

Brodie D and Schluger NW. 2005. The diagnosis of tuberculosis. Clin Chest

Med, 26: 247-471.

Brooks GF, Butel JS, and Morse SA. 1998. Mycobacterium. In: Jawetz,

Mehnick & Adelberg’s. Medical microbiology, pp: 279-288. Appleton &

Lange, Twenty frist edition, UK.

Buijtels PC and Petit PL. 2005. Comparison of NaOH-N-acetyl cysteine and

sulfuric acid decontamination methods for recovery of Mycobacteria from

clinical specimens. J Microbiol Methods, 62: 83-88.

Bulbuloglu E, Ciralik H, Okur E, Ozdemir G, Ezberci F and Cetinkaya A.

2006. Tuberculosis of the thyroid gland: review of the literature. World J

Surg, 30:149-155.

Burgess LJ, Maritz FJ and Le Roux I. 1995. Use of adenosine deaminase as a

diagnostic tool for tuberculous pleurisy. Thorax, 50: 672-674.

Butler WR, Kenneth CJ and Kilburn J. 1991. Identification of

Mycobacterium by High - performance liquid chromatography. J Clin

Microbiol, 29: 2468-2472.

BY Lee, Hefta SA and Brennan PJ. 1992. Characterization of the major

membrane protein of virulent Mycobacterium tuberculosis Infect Immun,

60: 2066-2074.

References

141

Cagatay AA, Caliskan Y, Aksoz S, Gulec L, Kucukoglu S, Cagatay Y, Berk

H, Ozsut H, Eraksoy H and Calangu S. 2004. Extra-pulmonary

tuberculosis in immunocompetent adults. Scand J Infect Dis, 36: 799-806.

Cambau E, Wichlacz C, Truffot-Pernot C and Jarlier V 1999. Evaluation of

the new MB redox system for detection of growth of Mycobacteria. J Clin

Microbiol, 37: 2013-2015.

Castro CC, Barros NG, Campos ZM and Cerri GG. 1995. CT scans of

cranial tuberculosis. Radiol Clin North Am, 33: 753-769.

Caviedes L, Lee TS, Gilman RH, Sheen P, Spellman E, Lee EH, Berg DE

and Montenegro-James S. 2000. Rapid, efficient detection and drug

susceptibility testing of Mycobacterium tuberculosis in sputum by

microscopic observation of broth cultures. J Clin Microbiol, 38: 1203-1208.

Chan ED, Heifets L and Iseman MD. 2000. Immunologic diagnosis of

tuberculosis. A review. Tuberc Lung Dis, 80: 131-140.

Chavhan GB, Hira P, Rathod K, Zacharia TT, Chawla A, Badhe P and

Parmar H. 2004. Female genital tuberculosis: hysterosalpingographic

appearances. Br J Radiol, 77: 164-169.

Chawla TC, Sharma A, Kiran U, Bhargava DK and Tandon BN. 1986.

Serodiagnosis of intestinal tuberculosis by enzyme immunoassay and

soluble antigen fluorescent antibody tests using a saline extracted antigen.

Tubercle, 67: 55-60.

Chedore P, Broukhanski G, Shainhouse Z, Jamieson F. 2006. False-Positive

amplified Mycobacterium Tuberculosis direct test results for samples

containing Mycobacterium leprae. J Clin Microbiol, 44: 612-613.

References

142

Cheebrough M. 2000. Medical laboratory manual for tropical countries, vol.

(2): Microbiology. PP: 70-99. Blackwell Scientific Publications, Oxford.

Chen HJ, Chiu NC, Kao HA and Wang TY. 2004. Perinatal tuberculosis in a

three-month-old infant. J Formos Med Assoc, 103: 144-147.

Chernousova LN, Suranova ED, Kalinina OA, Kulikovskaia NV, Kapina

MA, Dem'ianenko NV and Sergeeva LV. 1995. Affinity isolation of

mycobacterial antigens on monoclonal antibodies. Probl Tuberk, 2: 39-42.

Cho SN, Shin JS, Kim JD and Chong Y. 1990. Production of monoclonal

antibodies to lipoarabinomannan-B and use in the detection of

mycobacterial antigens in sputum. Yonsei Med J, 31: 333-338.

Cho, SN, Shin JS, Daffe M, Chong Y, Kim SK, and Kim JD. 1992.

Production of monoclonal antibody to a phenolic glycolipid of

Mycobacterium tuberculosis and its use in detection of the antigen in

clinical isolates. J Clin Microbiol, 30: 3065–3069.

Coker R, McKee M, Atun R, Dimitrova B, Dodonova E, Kuznetsov S and

Drobniewski F. 2006. Risk factors for pulmonary tuberculosis in Russia:

case-control study. BMJ, 14: 332 :85-87.

Collins DM, Skou B, White S, Bassett S, Collins L, For R, Hurr K, Hotter G

and de Lisle GW. 2005. Generation of attenuated Mycobacterium bovis

strains by signature-tagged mutagenesis for discovery of novel vaccine

candidates. Infect Immun, 73: 2379-2386.

Comstock GW, Daniel TM, Snider DE, Edwards PQ, Hopewell PC and

Vandiviere H M. 1981. The tuberculin skin test. Am Rev Respir Dis, 124:

356-363.

References

143

Cosivi O, Grange JM, Daborn CJ, Raviglione MC, Fujikura T and Cousins

D. 1998. Zoonotic tuberculosis due to Mycobacterium bovis in developing

countries. Emerg Infect Dis, 4: 1–14.

Costa JG, Santos AC, Rodrigues LC, Barreto ML and Roberts JA. 2005.

Tuberculosis in Salvador, Brazil: costs to health system and families. Rev

Saude Publica, 39: 122-128.

Crubezy E, Ludes B, Poveda JD, Clayton J, Crouau-Roy B and Montagnon

D. 1998. Identification of Mycobacterium DNA in an Egyptian Pott's

disease of 5,400 years old. CR Acad Sci III, 321: 941-951.

Crum NF. 2003. Tuberculosis presenting as epitrochlear lymphadenitis. Scand J

Infect Dis, 35 : 888-890.

Cummings PJ, Rowland SS, Hooper NE and Schwalbe RS. 1996.

Differential expression of a Mycobacterium tuberculosis protein recognized

by a mouse monoclonal antibody. Microbiol Immunol, 40: 883-886.

Daffe M and Etienne G. 1999. The capsule of Mycobacterium tuberculosis and

its implication for pathogenicty. Tubercle and lung disease, 79: 153- 169.

Dam P, Shirazee HH, Goswami SK, Ghosh S, Ganesh A, Chaudhury K and

Chakravarty B. 2006. Role of latent genital tuberculosis in repeated IVF

failure in the indian clinical setting. Gynecol Obstet Invest, 13;61: 223-227.

Daniel TM and Anderson PA. 1978. The isolation by immunoabsorbent

affinity chromatography and physicochemical characterization of

Mycobacterium tuberculosis antigen 5. Am Rev Respir Dis, 117: 533-539.

Daniel TM, Demurillo GL, Sawer JA, Friffin AM, Pint E, Debanne SM,

Espinosa P and Cespedes E. 1988. Field evaluation of enzyme linked

immunosorbent assay for serodiagnosis of tuberculosis. Am Rev Res Dis,

134: 662-665.

References

144

Davies PD and Yew WW. 2003. Recent developments in the treatment of

tuberculosis. Expert Opin Investig Drugs, 12: 1297-1312.

De Jonge N, Fillie YE and Deelder, AM. 1987. A simple and rapid treatment

(tricholoroacetic acid precipitation) of serum samples to prevent non-

specific reactions in the immunoassay of a proteoglycan. J Immunol Meth,

99: 195- 197.

Del Prete R, Picca V, Mosca AD Alagni M and Miragllotta G. 1998.

Detection of anti-lipoarabinomannan antibodies for the diagnosis of active

tuberculosis. Int J Tuberc Lung Dis, 2:160-163.

Delacourt C, Mani TM and Bonnerot V. 1993. Computed tomography with

normal chest radiograph in tuberculous infection. Arch Dis Child, 69: 430-

432.

Deodhar L, Gogate A, Padhi RC and Desai CR. 1998. Standardization of a

dot blot immunoassay for antigen detection in cases of pulmonary

tuberculosis & its evaluation with respect to the conventional techniques.

Indian J Med Res,108: 75-79.

Deshpande RG, Khan MB, Bhat DA and Navalkar RG. 1994. Skin

reactivity and fibronectin-binding property of TB66 (66-kDa protein of

Mycobacterium tuberculosis. J Med Microbiol, 41: 378-383.

Deshpande RG, Khan MB, Bhat DA and Navalkar RG. 1996. Isolation of a

33-kDa protein antigen from delipidified Mycobacterium tuberculosis

H37Rv. Med Microbiol Immunol, 185: 153-155.

Dietrich J, Weldingh K and Andersen P. 2006. Prospects for a novel vaccine

against tuberculosis. Vet Microbiol, 25:163-169.

References

145

Dochviri TZ, Salakaia AI, Katsitadze VA and Chigogidze TG. 2005.

Sensitivity of PCR method for detection of Mycobacteria tuberculosis in

patients with lung tuberculosis. Georgian Med News, 118: 14-17.

Donald PR, Schaaf HS and Schoeman JF. 2005. Tuberculous meningitis and

miliary tuberculosis: the Rich focus revisited. J Infect, 50 : 193-195.

Doskeiand SO and Berdai BP. 1980. Bacterial antigens detection in body

fluids: methods for rapid antigen concentration and reduction of nonspecific

reactions. J Clin Microbiol, 11: 380 – 384.

Dunbar BS. 1990. Two dimensional electrophoresis and immunological

techniques. pp:102-105. Plenim pren. New York and London.

Dursun B, Guler M, Budak K, Ceylan O and Atas E. 2003. Pott's disease and

different clinical presentations Pott's disease and different clinical

presentations. Tuberk Toraks, 51: 416-423.

Dye CS, Scheele R, Dolin G, Pathania, and Raviglione M. 1999. Global

burden of tuberculosis. Estimated incidence, prevalence, and mortality by

country. JAMA, 282:677–686.

Eintracht S, Silber E and Sonnenberg P. 2000. Analysis of adenosine

deaminase isoenzyme-2 (ADA-2) in cerebrospinal fluid in the diagnosis of

TB meningitis. J Neurol Neurosurg Psychiatry, 69: 137-138.

Elmoghazy E. 1997. National tuberculosis program report, epidemiological

review and action plan. Egyptian Ministry of Health, Cairo, Egypt.

Eltringham IJ, Drobniewski FA, Mangan JA, Butcher PD and Wilson SM.

1999. Evaluation of reverse transcription-PCR and a bacteriophage-based

assay for rapid phenotypic detection of rifampin resistance in clinical

isolates of Mycobacterium tuberculosis. J Clin Microbiol, 37: 3524-3527.

References

146

Engin G and Balk E. 2005. Imaging Findings of Intestinal Tuberculosis. J

Comput Assist Tomogr, 29: 37-41.

Espitia C and Mancilla R. 1989. Identification, isolation and partial

characterization of Mycobacterium tuberculosis glycoprotein antigens. Clin

Exp Immunol,77: 378-383.

Fenniche S, Ben Jennet S, Marrak H, Khayat O, Zghal M, Ben Ayed M and

Mokhtar I. 2003. Cutaneous tuberculosis: anatomoclinical and evaluative

features (26 cases). Ann Dermatol Venereol, 30: 1021-1024.

Fernandez CM, Perez BS and Ledo L. 1991. Ascites adenosine deaminase

activity is decreased in tuberculous ascites with low protein content. Am J

Gastroenterol, 86: 1500-1503.

Fernandez JM, Alonso ME, Lobato DL and Martinez SJ. 1995. Extra-

pulmonary tuberculosis: retrospective study of 107 cases. An Med Interna.,

12: 212-215.

Flament SM and Perronne C. 1997. The natural history of tuberculosis

infection and skin tuberculin reaction. Rev Mal Respir, 14: 27-32.

Freer G, Florio W, Dalla Casa B, Castagna B, Maisetta G, Batoni G,

Corsini V, Senesi S and Campa M. 1998. Characterization of antigens

recognized by new monoclonal antibodies raised against culture filtrate

proteins of Mycobacterium bovis bacillus Calmette-Guerin. FEMS

Immunol Med Microbiol, 20: 129-138.

Fujita Y, Doi T, Maekura R, Ito M and Yano I. 2006. Differences in

serological responses to specific glycopeptidolipid-core and common lipid

antigens in patients with pulmonary disease due to Mycobacterium

tuberculosis and Mycobacterium avium complex. J Med Microbiol, 55

:189-199.

References

147

Gagneux S, Deriemer K, Van T, Kato-Maeda M, de Jong BC, Narayanan S,

Nicol M, Niemann S, Kremer K, Gutierrez MC, Hilty M, Hopewell PC,

Small PM. 2006. Variable host-pathogen compatibility in Mycobacterium

tuberculosis. Proc Natl Acad Sci U S A, 103 : 2869-2873.

Gambbir IS, Mehta M, Singh DS and Khanna HD. 1999. Evaluation of

CSF-adenosine deaminase activity in tubercular meningitis. J Assoc

Physician India, 47:192-194.

Garfin GE. 1990. One dimensional gel electrophoresis. In: Duetscher MP (edt).

Guide to protein purification. PP: 425-428. Academic press Inc., San

Diago, California.

Garg SK, Tiwari RP, Tiwari D, Singh R, Malhotra D, Ramnani VK, Prasad

GB, Chandra R, Fraziano M, Colizzi V and Bisen PS. 2003. Diagnosis

of tuberculosis: available technologies, limitations, and possibilities. J Clin

Lab Anal, 17: 155-163.

Gebre-Selassie S. 2003. Evaluation of the concentration sputum smears

technique for the laboratory diagnosis of pulmonary tuberculosis. Trop

Doct, 33: 160-162.

Gibson MS, Puckett ML and Shelly ME. 2004. Renal tuberculosis.

Radiographics, 24: 251-256.

Gomes M, Saad R and Stirbulov R. 2003. Pulmonary tuberculosis:

relationship between sputum bacilloscopy and radiological lesions. Rev Inst

Med Trop Sao Paulo, 45: 275-281.

Gomez JE and McKinney JD. 2004. Tuberculosis persistence, latency, and

drug tolerance. Tuberculosis, 84: 29-44.

Gradmann C. 2006. Robert Koch and the white death: from tuberculosis to

tuberculin. Microbes Infect, 8 :294-301.

References

148

Grange JM. 2002. Mycobacterium. In: Greenwood D, Slack R and Peutheres

(edts). Medical microbiology.16 th ed. pp:200-214. Churchill Livingstone,

UK.

Grassi M, Volpe E, Colizzi V and Mariani F. 2006. An improved, real-time

PCR assay for the detection of GC-rich and low abundance templates of

Mycobacterium tuberculosis. J Microbiol Methods, 64: 406-410

Gülgün E M, Bülent AM, Gülden AM and Mehtap TM. 2000. Imaging of

Extra-pulmonary Tuberculosis. Radiographics, 20:471-488.

Gupta S, Kumari S and Banwalikar JN. 1995. Diagnostic utility of the

estimation of mycobacterial antigen A60 specific immunoglobulins IgM,

IgA and IgG in the sera of cases of adult human tuberculosis. Tubercle

Lung Dis, 76: 418-424.

Haile M and Kallenius G. 2005. Recent developments in tuberculosis

vaccines. Curr Opin Infect Dis, 18: 211-215.

Hanna BA, Ebrahimzadeh A, Elliott LB, Morgan MA, Novak SM, Rusch-

Gerdes S, Acio M, Dunbar DF, Holmes TM, Rexer CH, Savthyakumar

C and Vannier AM. 1999. Multi-center evaluation of the BACTEC

MGIT 960 system for recovery of Mycobacteria. J Clin Microbiol, 37: 748-

752.

Harboe M, Das AK, Mitra D, Ulvund G, Ahmad S, Harkness RE, Das D,

Mustafa AS and Wiker HG. 2004. Immunodominant B-cell epitope in

the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis

cross-reacting with glutathione S-transferase. Scand J Immunol, 59: 190-

197.

Hayati IN, Ismail Y and Zurkurnain Y. 1993. Extra-pulmonary

tuberculosis: a two-year review of cases at the General Hospital Kota

Bharu. Med J Malaysia, 48: 416-420.

References

149

Heifets L, Linder T, Sanchez T, Spencer D and Brennan J. 2000. Two liquid

medium systems, Mycobacteria growth indicator tube and MB redox tube,

for Mycobacterium tuberculosis isolation from sputum specimens. J Clin

Microbiol, 38: 1227-1230.

Hiraki A, Aoe K, Eda R, Maeda T, Murakami T, Sugi K and Takeyama H.

2004. Comparison of six biological markers for the diagnosis of

tuberculous pleuritis. Chest, 125: 987-989.

Houben EN, Nguyen L, Pieters J. 2006. Interaction of pathogenic

Mycobacteria with the host immune system. Curr Opin Microbiol, 9:

76-85.

Hu G, Lin G, Wang M, Dick L, Xu RM, Nathan C and Li H. 2006.

Structure of the Mycobacterium tuberculosis proteasome and mechanism of

inhibition by a peptidyl boronate. Mol Microbiol, 59: 1417-1428.

Huang RR. 1990. Purified protein derivative antigen purified by using a

monoclonal antibody to Mycobacterium tuberculosis. Zhonghua Jie He Hu

Xi Za Zhi, 13: 289-291.

Humphrey DM and Weiner MH. 1987. Mycobacterial antigen detection by

immunohistochemistry in pulmonary tuberculosis. Hum Pathol, 18: 701-

708.

Humphries MJ and Lam WK. 1998. Non-respiratory tuberculosis. In:

Clinical Tuberculosis (2nd Edition), Davies PD (edt), pp: 188-189.

Chapman and Hall, London.

Hussain SF, Irfan M, Abbasi M, Anwer SS, Davidson S, Haqqee R, Khan

JA and Islam M. 2004. Clinical characteristics of 110 miliary tuberculosis

References

150

patients from a low HIV prevalence country. Int J Tuberc Lung Dis, 8: 493-

499.

Hwang S, Simsir A, Waisman J and Moreira AL. 2004. Extra-pulmonary

tuberculosis as a mimicker of neoplasia. Diagn Cytopathol, 30: 82-87.

Ismail Y and Muhamad A. 2003. Protean manifestations of gastrointestinal

tuberculosis. Med J Malaysia, 58: 345-349.

Jackkett PS, Bothamley GH, Batra HV, Mistry A , Young DH and Ivanyi

J. 1988. Specificity of antibodies to immunodominaant Mycobacterial

antigens in pulmonary tuberculosis. J Clin Microbiol, 26: 2313-2318.

Jones-Lopez EC, Okwera A, Mayanja-Kizza H, Ellner JJ, Mugerwa RD

and Whalen CC. 2006. Delayed –type hypersensitivity skin test reactivity

and survival in HIV infected patients in Uganda: should anergy be a start

antiretroviral therapy in low-income countries ? Am J Trop Med Hyg, 74

:154-161.

Julius MC and Robert EL. 2000. Atlas of immunology. PP:128. CRC press.

USA.

Jutte P and Van Loenhout-Rooyackers J. 2006. Routine surgery in addition

to chemotherapy for treating spinal tuberculosis. Cochrane Database Syst

Rev, 25 :CD004532.

Kadival GV, Chaparas SD and Hussong D. 1987. Characterization of

serologic and cell-mediated reactivity of a 38-kDa antigen isolated from

Mycobacterium tuberculosis. J Immunol, 139: 2447- 24451.

Kansal R and Khuller GK. 1991. Detection of mannophosphoinositide

antigens in sputum of tuberculosis patients by dot enzyme immunoassay.

Med Microbiol Immunol, 180: 73-78.

References

151

Kapoor VK. 1998. Abdominal tuberculosis : the Indian contribution. Indian J

Gastroenterol, 17 : 141-147.

Karen MD, Kay HK, Patrick JB, and Johnn TB. 1995. Evidence for

Glycosylation sites on the 45-kilodalton glycoprotein of Mycobacterium

tuberculosis. Infect Immun, 63: 2846–2853.

Kart L, Akduman D, Altin R, Tor M, Unalacak M, Begendik F, Erdem F

and Alparslan U. 2003. Fourteen-year trend of tuberculosis dynamics in

the northwest of Turkey. Respiration, 70: 468-474.

Kashyap RS, Dobos KM, Belisle JT, Purohit HJ, Chandak NH, Taori GM

and Daginawala HF. 2005. Demonstration of components of antigen 85

complex in cerebrospinal fluid of tuberculous meningitis patients. Clin

Diagn Lab Immunol, 12: 752-758.

Kashyap RS, Kainthla RP, Biswas SK, Agarwal N, Chandak NH, Purohit

HJ, Taori GM and Daginawala HF. 2003. Rapid diagnosis of tuberculous

meningitis using the Simple Dot ELISA method. Med Sci Monit, 9: 123-

126.

Kathleen AA, Pleydell E, Williams MC, Lane EP, Nyange JF, and Michel

AL. 2002. Mycobacterium tuberculosis: An Emerging Disease of Free-

Ranging Wildlife. Emerging Infect Dis, 8: 599-601.

Katti MK. 2001. Immunodiagnosis of tuberculous meningitis: rapid detection

of mycobacterial antigens in cerebrospinal fluid by reverse passive

haemagglutination assay and their characterization by Western blotting.

FEMS Immunol Med Microbiol, 31: 59-64.

Kehinde AO, Obaseki FA, Cadmus SI and Bakare RA. 2005. Diagnosis of

tuberculosis: urgent need to strengthen laboratory services. J Natl Med

Assoc, 97: 394-396.

References

152

Khomenko AG, Bayensky AV, Chernousova LN, Kulikovskaya NV,

Demianenko NV and Litvinov VI. 1996. Serodiagnosis of tuberculosis:

detection of Mycobacterial antibodies and antigens. Tuber Lung Dis, 77:

510-515.

Khubnani H and Munjal K. 2005. Application of bleach method in diagnosis

of extra-pulmonary tuberculosis. Indian J Pathol Microbiol, 48: 546-550.

Kidane D, Olobo JO, Habte A, Negesse Y, Aseffa A, Abate G, Yassin MA,

Bereda K and Harboe M. 2002. Identification of the causative organism

of tuberculous lymphadenitis in Ethiopia by PCR. J Clin Microbiol, 40:

4230-4234.

Kim SC, Kang SI, Kim DW, Kim SC, Cho SN, Hwang JH, Kim Y, Song SD

and Kim YH. 2003. Development and evaluation of an automated stainer

for acid-fast bacilli. Med Eng Phys, 25: 341-347.

Koch R. 1882. Aetiologie der tuberculose. Berlin. Klin. Wochenschr,19: 221-

230.

Kohler G and Milstein C. 1975. Derivation of specific Ab - producing tissue

culture and tumor lines by cell fusion. Eur J Immunol, 6: 511 - 514.

Kolk A, Klatser PR, Eggelte TA, Kuijper S, Jonge S and Leeuwen J. 1984.

Production and characterization of monoclonal antibodies to

Mycobacterium tuberculosis, M. bovis (BCG) and M. leprae. Clin Exp

Immunol, 58: 511-521.

Kulchavenya E and Khomyakov V. 2006. Male genital tuberculosis in

Siberians. World J Urol, 21:1-5.

Kulkarni SP, Jaleel MA and Kadival GV. 2005. Evaluation of an in-house-

developed PCR for the diagnosis of tuberculous meningitis in Indian

children. J Med Microbiol, 54: 369-373.

References

153

Kumar KS, Raja A, Devi KR and Paranjape RS. 2000.

Production & characterization of monoclonal antibodies to Mycobacterium

tuberculosis. Indian J Med Res, 112: 37-46.

Lado FL, Tunez B V, Golpe AL and Ferreiro MJ and Cabarcos OA. 2000.

Extra-pulmonary tuberculosis in our area. Forms of presentation. An Med

Interna, 7: 637-641.

Laemmli G. 1970. Cleavage of structural proteins during assembly of the need

of the bacteriophage T4. Nature, 277: 680-685.

Laidlaw M. 1989. Mycobacterium: tubercle bacilli. In: Collee JG, Duguid JP,

Fraser A.G, Marmion BP (eds). Mackie & McCartney, Practical medical

microbiology, vol. 2: PP 399-416, 13th ed. Churchill Livingstone,

Edinburgh.

Lalit K. 2004. Extra-pulmonary tuberculosis: Coming out of the shadows.

Indian J Tuberc, 51: 189-190.

Langermans JA, Doherty TM, Vervenne RA, van der Laan T, Lyashchenko

K, Greenwald R, Agger EM, Aagaard C, Weiler H, van Soolingen D,

Dalemans W, Thomas AW and Andersen P. 2005. Protection of

macaques against Mycobacterium tuberculosis infection by a subunit

vaccine based on a fusion protein of antigen 85B and ESAT-6. Vaccine, 15:

2740-2750.

Lenka MP, Arias B, Lan NN, LY MH, Sjoukje K H, Jansen and Arend

HJ. 2000. Development of antigen detection assay for diagnosis of

tuberculosis using sputum samples. J Clin Microbiol,38: 2278–2283.

Liberato IR, de Albuquerque Mde F, Campelo AR and de Melo HR. 2004.

Characteristics of pulmonary tuberculosis in HIV seropositive and

seronegative patients in a Northeastern region of Brazil. Rev Soc Bras Med

Trop,37: 46-50.

References

154

Liu JJ, Yao HY and Liu EY 2005. Analysis of factors affecting the

epidemiology of tuberculosis in China. Int J Tuberc Lung Dis, 9: 450-454.

Liu JJ, Yao HY, Liu EY. 2004. Relationship between tuberculosis prevalence

and socio-economic factors in China. Zhonghua Liu Xing Bing Xue Za Zhi,

25: 1032-1034.

Liu YC, Huang TS and Huang WK. 1999. Comparison of a non-radiometric

liquid-medium method (MB REDOX) with the BACTEC system for

growth and identification of Mycobacteria in clinical specimens. J Clin

Microbiol ,37: 4048-4050.

Lowry OH, Rosenbrough NJ, Farr AL and Randall RJ. 1951. Protein

measurement with folin - phenol reagent. J Biol Chem, 193: 265 - 275.

Luo D. 1990. Immunohistochemical demonstration of mycobacterial antigen.

Zhonghua Jie He Hu Xi Za Zhi, 13: 360-2, 381-382.

Makiyama A, Okuyama Y, Okajima T and Fujimoto S. 2003.

Tuberculous peritonitis. J Gastroenterol, 38: 1167-1170.

Manos J and Wagner GE. 1997. Mycobacteria, In: Microbiology and

infectious diseases. Gabriel, V., chapter 18, pp:167 - 175. Williams &

Wilkins, 3 rd edition. London.

Marais BJ, Wright CA, Schaaf HS, Gie RP, Hesseling AC, Enarson DA and

Beyers N. 2006. Tuberculous lymphadenitis as a cause of persistent

cervical lymphadenopathy in children from a tuberculosis-endemic area.

Pediatr Infect Dis J, 25:142-146.

Mark D and Perkins MD. 2000. New diagnostic tools for tuberculosis.

International Journal of Tuberculosis and Lung Disease, 4: 182-188.

References

155

Marmor M, Parnes N and Dekel S. 2004. Tuberculosis infection complicating

total knee arthroplasty: report of 3 cases and review of the literature. J

Arthroplasty, 19: 397-400.

Martin G and Lazarus A. 2000. Epidemiology and diagnosis of tuberculosis:

recognition of at-risk patients is key to prompt detection. Postgrad Med,

108 : 42-54.

Martin E M, Sandra MA, Inger B, Ottenhoff H M and Peter A. 2001. Use of

ESAT-6 and CFP-10 antigens for diagnosis of extra-pulmonary

tuberculosis. J Infect Dis, 183: 175-176.

Mathai A, Radhakrishnan VV, George SM and Sarada C. 2001. A newer

approach for the laboratory diagnosis of tuberculous meningitis. Diagn

Microbiol Infect Dis, 39: 225-228.

Mathai A, Radhakrishnan VV, Sarada C and George SM. 2003. Detection

of heat stable Mycobacterial antigen in cerebrospinal fluid by Dot-

Immunobinding assay. Neurol India, 51: 52-54.

Matthew JF and Mary WV. 1996. Immunopathology of tuberculosis: roles of

macrophages and monocytes. Infect Immu, 64: 683–690.

Middleton AM, Chadwick MV, Nicholson AG, Wilson R, Thornton DJ,

Kirkham S and Sheehan JK. 2004. Interaction between Mycobacteria

and mucus on a human respiratory tissue organ culture model with an air

interface. Exp Lung Res, 30:17-29.

Mishra OP, Yusaf S, Ali Z, Nath G and Das BK. 2000. Adenosine

deaminase activity and lysozyme levels in children with tuberculosis. J

Trop Pediatr, 46:175-178.

Mitchinson, MJ, Akno J, Edwards, WA, Lepage RW, and Minson AC.

1996. Essential of pathology, chapter 8, pp:137-151, Black well Science,

London.

References

156

Mitchison DA. 2005. The control of tuberculosis: Progress & prospect. Indian J

Med Res, 121:137-139.

Morse D, Brothwell DR and Uckop J. 1964. Tuberculosis in ancient Egypt.

Am Rev Dis., 93: 524 - 530.

Murray SJ, Barrett A, Magee JG and Freeman R. 2003. Optimization of acid

fast smears for the direct detection of Mycobacteria in clinical samples. J

Clin Pathol, 56: 613-615.

Nahid P, Pai M and Hopewell PC. 2006. Advances in the diagnosis and

treatment of tuberculosis. Proc Am Thorac Soc, 3: 103-110.

Nair RE, Banerjee S, Kumar S and Harinath BC. 2000. Isolation and

characterization of a 31 kDa mycobacterial antigen from tuberculous sera

and its identification with in vitro released culture filtrate antigen of mtb

H37Ra bacilli. Scand J Infect Dis, 32: 551-556.

Nakamura RM, Velmonte MA, Kawajiri K, Ang CF, Frias RA, Mendoza,

MT, Montoya JC, Honda I, Haga S and Toida I. 1998. MPB 64

mycobacterial antigen: A new skin-test reagent through patch method for

rapid diagnosis of active tuberculosis. Int J Tuber Lung Dis, 2: 541-546.

Nalini B and Vinayak S. 2006. Tuberculosis in ear, nose, and throat practice:

its presentation and diagnosis. Am J Otolaryngol, 27: 39-45.

Narang P, Narang R, Narang R, Mendiratta DK, Sharma SM and Tyagi

NK. 2005. Prevalence of tuberculous lymphadenitis in children in Wardha

district, Maharashtra State, India. Int J Tuberc Lung Dis, 9: 188-194.

Nayar RC, Al Kaabi J and Ghorpade K. 2004. Primary nasal tuberculosis: a

case report. Ear Nose Throat J, 83:188-191.

References

157

N'dri Oka D, Varlet G, Cowppli-Bony P, Haidara A and Ba Zeze V. 2004.

Diagnosis and treatment of extensive vertebral tuberculosis. Rev Neurol,

160: 419-423.

Ng TT, Strang J I and Wilkins E G. 1995. Serodiagnosis of pericardial

tuberculosis. Quarterly J Med, 88: 317-320.

Nissapatorn V, Kuppusamy I, Rohela M, Anuar AK and Fong MY. 2004.

Extra-pulmonary tuberculosis in Peninsular Malaysia: retrospective study

of 195 cases. Southeast Asian J Trop Med Public Health, 35: 39-45.

Okolo SN, Nwana EJ and Mohammed AZ. 2003. Histopathologic diagnoses

of lymphadenopathy in children in Jos, Nigeria. Niger Postgrad Med J, 10:

165-167.

Oktay MF, Topcu I, Senyigit A, Bilici A, Arslan A, Cureoglu S and Yildirim

M. 2006. Follow-up results in tuberculous cervical lymphadenitis. J

Laryngol Otol, 120:129-132.

Padayatchi N, Bamber S, Dawood H and Bobat R. 2006. Multidrug-resistant

tuberculous meningitis in children in Durban, South Africa. Pediatr Infect

Dis J, 25:147-150.

Parisi MT, Jensen MC and Wood BP. 1994. Pictorial review of the usual and

unusual roentgen manifestations of childhood tuberculosis. Clin Imag, 18:

149-154.

Park JK, Lee SH, Kim SG, Kim HY, Lee JH, Shim JH, Kim JS, Jung HC

and Song IS. 2005. A case of intestinal tuberculosis presenting massive

hematochezia controlled by endoscopic coagulation therapy. Korean J

Gastroenterol, 45: 60-63.

Parthasarathy A. 2003. Controversies in BCG immunization. Indian J Pediatr,

70: 585-586.

References

158

Pereira Arias-Bouda LM, Nguyen LN, Ho LM, Kuijper S, Jansen HM and

Kolk AH. 2000. Development of antigen detection assay for diagnosis of

tuberculosis using sputum samples. Clin Microbiol,38: 2278-2283.

Petterson T, Klockars M, Weber TH and Somer H. 1992. Diagnostic value

of cerebrospinal fluid adenosine deaminase determination. Scand J Infect

Dis, 24: 121-122.

Pfyffer GE. 1999. Nucleic acid amplification for mycobacterial diagnosis. J

Infect, 39: 21-26.

Philip WB. 2003. Laboratory role in tuberculosis control. WMJ,102:31-34.

Rabaud C, Engozogho M, Dailloux M, Hoen B, May T and Canton P. 1997.

Tuberculosis in Lorraine, France: study of prognostic factors. Int J Tuberc

Lung Dis, 1: 246-249.

Radhakrishnan VV and Mathai A. 1991. A dot-Immunobinding assay for the

laboratory diagnosis of tuberculous meningitis and its comparison with

enzyme-linked immunosorbent assay. J Appl Bacteriol, 71: 428-33.

Radhakrishnan VV, Sehgal S and Mathai A. 1990. Correlation between

culture of Mycobacterium tuberculosis and detection of mycobacterial

antigens in cerebrospinal fluid of patients with tuberculous meningitis. J

Med Microbiol, 33: 223-226.

Rahman M, Sekimoto M, Takamatsu I, Hira K, Shimbo T and Toyoshima

K. 2001. Economic evaluation of universal BCG vaccination of Japanese

infants. Inter J Epidemiol, 30: 380-385.

Rajpal SK, Rani PK, Saibal KB, Neha AN, Chandak HJ, Purohit GM, ,

Hatim FD. 2003. Rapid diagnosis of tuberculous meningitis using the

simple dot ELISA method . Med Sci Monit, 9: 123-126.

References

159

Ramachandran R and Paramasivan CN. 2003. What is new in the diagnosis

of tuberculosis. Techniques for diagnosis of tuberculosis. Ind J Tub, 50:

133-141.

Rasolofo V and Chanteau S. 1999. Field evaluation of rapid tests for

tuberculosis diagnosis. J Clin Microbiol, 37: 4201-4202.

Ratanasuwan W, Kreiss JK, Nolan CM, Schaeffler BA and Suwanagool S,

Tunsupasawasdikul S. 1997. Evaluation of the Myco-Dot test for the

diagnosis of tuberculosis in HIV seropositive and seronegative patients. Int

J Tuberc Lung Dis, 1: 259- 264.

Raut AA, Nagar AM, Muzumdar D, Chawla AJ, Narlawar RS, Fattepurkar

S and Bhatgadde VL. 2004. Imaging features of calvarial tuberculosis: a

study of 42 cases. AJNR Am J Neuroradiol, 25: 409-414.

Reinout VC, Tom HM, and Jos WM. 2002. Innate Immunity to

Mycobacterium tuberculosis. Clin Microbio Rev, 15: 294–309.

Reuter H, Burgess LJ, Schneider J, Van Vuuren W and Doubell AF. 2006.

The role of histopathology in establishing the diagnosis of tuberculous

pericardial effusions in the presence of HIV. Histopathology, 48:295-302.

Roth A, Schaberg T and Mauch H. 1997. Molecular diagnosis of tuberculosis:

current clinical validity and future perspectives. Eur Respir J, 10: 1877-

1891.

Sabetay C, Enache D, Plesea E, Zaharia B, Stoica A, Rosca A, Singer I,

Farcas I and Malos A. 2000. Intestinal tuberculosis in children.

Differential diagnosis and treatment. Chirurgia, 95: 179-191.

Sada E, Aguilar D, Torres M and Herrera T. 1992. Detection of

lipoarabinomannan as a diagnostic test for tuberculosis. J Clin Microbiol,

30: 2415-2418.

References

160

Said A, Rashed HG, Morlock GP, Woodley CL, El Shanawy O, and

Cooksey RC. 2001. Characterization of IS6110 restriction fragment length

polymorphism patterns and mechanisms of antimicrobial resistance for

multidrug-resistant isolates of Mycobacterium tuberculosis from a major

reference hospital in Assiut, Egypt. J Clin Microbiol, 39: 2330-2334.

Salata RA, Sanson AJ, Malhotra IJ, Wiker HG, Harboe M, Phillips NB and

Daniel TM. 1991. Purification and characterization of the 30,000 dalton

native antigen of Mycobacterium tuberculosis and characterization of six

monoclonal antibodies reactive with a major epitope of this antigen. J Lab

Clin Med, 118: 589-598.

Samanich KM, Keen MA, Vissa VD, Harder JD, Spencer JS, Belisle JT,

Zolla-Pazner S and Laal S. 2000. Serodiagnostic potential of culture

filtrate antigens of Mycobacterium tuberculosis. Clin Diagn Lab Immunol,

7: 662-668.

Selvakumar N, Prabhakaran E, Murthy BN, Sivagamasundari S,

Vasanthan S, Govindaraju R, Perumal M, Wares F, Chauhan LS,

Santha T, Narayanan PR. 2005. Application of lot sampling of sputum

AFB smears for the assessment of microscopy centres. Int J Tuberc Lung

Dis, 9 :306-309.

Sengoz G. 2005. Evaluating 82 cases of tuberculous meningitis. Tuberk Toraks,

53: 51-56.

Sethi A, Sareen D, Sabherwal A, Malhotra V. 2006. Primary parotid

tuberculosis: varied clinical presentations. Oral Dis, 12:213-215.

Sharma MP and Bhatia V. 2004. Abdominal tuberculosis. Indian J Med

Res,120: 305-315.

References

161

Sharma SK and Banga A. 2005. Pleural fluid interferon-gamma and

adenosine deaminase levels in tuberculosis pleural effusion: a cost-

effectiveness analysis. J Clin Lab Anal,19: 40-46.

Shaw RJ and Taylor GM. 1998. Polymerase chain reaction: Applications for

diagnosis, drug sensitivity and strain identification of M. tuberculosis

complex. In: Clinical Tuberculosis (2nd Edition), Davies PD (edt), pp: 47.

Chapman and Hall, London,

Shimada A, Oshita Y, Kinoshita T, Rikimaru T and Aizawa H. 2003. A

case of primary nasopharyngeal tuberculosis. Kekkaku, 78: 751-755.

Simkus A. 2004. Thyroid tuberculosis. Medicina, 40: 201-204.

Somoskvِi A and Magyar P. 1999. Comparison of the Mycobacteria Growth

Indicator Tube with MB Redox, Lwِenstein-Jensen, and Middlebrook 7H11

Media for Recovery of Mycobacteria in Clinical Specimens. J Clin

Microbiol,37: 1366-1369.

Sood J, Gupta OP, Narang P, Cheirmaraj K, Reddy MV and Harinath BC.

1991. Penicillinase ELISA for detection of tubercular antigen in

tuberculosis. J Commun Dis, 23:173-177.

Srivastava KL, Bansal M, Gupta S, Srivastava R, Kapoor RK, Wakhlu I

and Srivastava BS. 1998. Diagnosis of tuberculous meningitis by

detection of antigen and antibodies in CSF and sera. Indian Pediatr, 35:

841-850.

Stavri H, Moldovan O, Mihaltan F, Banica D and Doyle RJ. 2003. Rapid dot

sputum and serum assay in pulmonary tuberculosis. J Microbiol Methods, 52

: 285-296.

References

162

Stott DI. 1989. Immunoblotting and dot blotting. J Immunol Methods, 119:

153-187.

Sumi M G, Mathai A, Sarada C, and Dhakrishnan VV. 1999. Rapid

diagnosis of tuberculous meningitis by a dot Immunobinding assay to

detect mycobacterial antigen in cerebrospinal fluid specimens. J Clin

Microbiol, 37: 3925–3927.

Sutlas PN, Unal A, Forta H, Senol S and Kirbas D. 2003. Tuberculous

meningitis in adults: review of 61 cases. Infection, 31:387-391.

Tekkel M, Rahu M, Loit HM, Baburin A. 2002. Risk factors for pulmonary

tuberculosis in Estonia. Int J Tuberc Lung Dis, 6: 887-894.

Theobald S, Tolhurst R and Squire SB. 2006. Gender, equity: new approaches

for effective management of communicable diseases. Trans R Soc Trop

Med Hyg, 100: 299-304.

Theodora F, Chrys C, Anthony J R, Antony B and Paul R W. 1991.

Purification and characterization of major antigens from a Mycobacterium

bovis culture filtrate. Infect Immun, 59: 800-807.

Thornton CG, MacLellan KM, Brink TL Jr and Passen S. 1998. In vitro

comparison of NALC-NaOH, tween 80, and C18-carboxypropylbetaine for

processing of specimens for recovery of Mycobacteria. J Clin Microbiol,

36: 3558-3566.

Thwaites GE, Chau TT and Farrar JJ. 2004. Improving the bacteriological

diagnosis of tuberculous meningitis. J Clin Microbiol, 42: 378-379.

Titov AG, Vyshnevskaya EB, Mazurenko SI, Santavirta S and Konttinen

YT. 2004. Use of polymerase chain reaction to diagnose tuberculous

References

163

arthritis from joint tissues and synovial fluid. Arch Pathol Lab Med, 128:

205-209.

Tiwari V, Jain A and Verma RK. 2003. Application of enzyme amplified

Mycobacterial DNA detection in the diagnosis of pulmonary & extra-

pulmonary tuberculosis. Indian J Med Res, 118: 224-228.

Towbin H, Staechlin T, and Gordan J. 1979. Electrophoretic transfer of

proteins from polyacrylamide gels to nitrocellulose sheets, procedures and

some application. National Academy of Sciences, USA, 76: 4350 - 4354.

Tsai MC, Chakravarty S, Zhu G, Xu J, Tanaka K, Koch C, Tufariello J,

Flynn J and Chan J. 2006. Characterization of the tuberculous granuloma

in murine and human lungs: cellular composition and relative tissue oxygen

tension. Cell Microbiol, 8: 218-232.

Tzoanopoulos D, Mimidis K, Giaglis S, Ritis K and Kartalis G. 2003. The

usefulness of PCR amplification of the IS6110 insertion element of M.

tuberculosis complex in ascetic fluid of patients with peritoneal

tuberculosis. Eur J Intern Med,14: 367-371.

Valdes L, Alvarez D and San Jose E. 1995. Tuberculous pleuritis: a study of

254 patients. Arch Intern Med, 158: 2017-2021.

Valdes L, San Jose E and Alvarez D 1993. Diagnosis of tuberculous pleurisy

using the biologic parameters adenosine deaminase, lysozyme and

interferon gamma. Chest, 103: 458-465.

Valdes L, San Jose E, Alvarez D and Valle JM. 1996. Adenosine deaminase

isoenzyme analysis in pleural effusions: diagnostic role, and relevance to

the origin of increased ADA in tuberculous pleurisy. Eur Respir J, 9: 797-

751.

References

164

Valerie AS, Howard NC and Margaraet MH. 2002. Mycobacterium

tuberculosis. In: Max S (edt). Molecular medical microbiology. PP:1731-

1743. Academic press. London.

Vardareli E, Kebapci M, Saricam T, Pasaoglu O and Acikalin M. 2004.

Tuberculous peritonitis of the wet ascetic type: clinical features and

diagnostic value of image-guided peritoneal biopsy. Dig Liver Dis, 36:199-

204.

Vasankari T, Liippo K and Tala E. 2003. Overt and cryptic miliary

tuberculosis misdiagnosed until autopsy. Scand J Infect Dis, 35: 794-796.

Villena V, Navarro- Gonzalvez JA and Garcia-Benayas C. 1996. Rapid

automated determination of adenosine deaminase and lysozyme for

differentiating tuberculous and non tuberculous pleural effusions. Clin

Chem, 42: 218-221.

Wade MM and Zhang Y. 2004. Mechanisms of drug resistance in

Mycobacterium tuberculosis. Front Biosci, 1 :975-994.

Wadee AA, Boting L, and Reddy SG. 1990. Antigen Capture Assay for

Detection of a 43-Kilodalton Mycobacterium tuberculosis antigen. J Clin

Microbiol, 28: 2786-2791.

Walsh A and McNerney R. 2004. Guidelines for establishing trials of new tests

to diagnose tuberculosis in endemic countries. Int J Tuberc Lung Dis, 8:

609-613.

Wan GH, Lu SC and Tsai YH. 2004. Polymerase chain reaction used for the

detection of airborne Mycobacterium tuberculosis in health care settings.

Am J Infect Control, 32: 17-22.

References

165

Wedlock DN, Denis M, Skinner MA, Koach J, de Lisle GW, Vordermeier

HM, Hewinson RG, van Drunen Littel-van den Hurk S, Babiuk LA,

Hecker R and Buddle BM. 2005. Vaccination of cattle with a CpG

oligodeoxynucleotide-formulated Mycobacterial protein vaccine and

Mycobacterium bovis BCG induces levels of protection against bovine

tuberculosis superior to those induced by vaccination with BCG alone.

Infect Immun, 73: 3540-3546.

Weldingh K and Andersen P. 1999. Immunological evaluation of novel

Mycobacterium tuberculosis culture filtrate proteins. FEMS Immunol Med

Microbiol, 23:159-164.

Whitelaw DA, Currie G and Littleton N. 2004. Disseminated tuberculous

osteitis. S Afr Med J, 94: 92.

Whiteman ML. 1997. Neuroimaging of central nervous system tuberculosis in

HIV-infected patients. Neuroimaging Clin North Am, 7:199-214.

WHO. 1986. Characterization of antigens recognized by Mycobacterium

specific monoclonal antibodies. Infec Immune, 51: 718.

WHO. 1997. WHO Tuberculosis Diagnostics Work-shop: product development

guidelines,1-27.

WHO. 2000. Global Tuberculosis Report, Geneva.

Williams A, Goonetilleke NP, McShane H, Clark SO, Hatch G, Gilbert SC

and Hill AV. 2005. Boosting with poxviruses enhances Mycobacterium

bovis BCG efficacy against tuberculosis in Guinea Pigs. Infect Immun, 73:

3814-3816.

Yanez MA, Coppola MP, Russo DA, Delaha E, Chaparas SD and Yeager

HJ. 1986. Determination of mycobacterial antigens in sputum by enzyme

immunoassay. J Clin Microbiol, 23:822-825.

References

166

Yano O, Fukuda T, Abe H and Sudo T. 1984. Purification and

characterization of a tuberculin-active substance from Mycobacterium bovis

BCG. J Biochem, 96 : 523-531.

Youssef FG, El-Sakka H, Azab A, Eloun S, Chapman GD, Ismail T,

Mansour H, Hallaj Z and Mahoney F. 2004. Etiology antimicrobial

susceptibility profiles, and mortality associated with bacterial meningitis

among children in Egypt. Ann Epidemiol, 14: 44-48.

Zengyi Chang , Todd PP, Joanita J, Irwin HL, Irina S, Wah C, Hiram

FG and Florante AQ. 1996. Mycobacterium tuberculosis 16-kDa

Antigen (Hsp16.3) functions as an oligomeric structure in vitro to suppress

thermal aggregation. J Biochem, 271: 7218-7223.

Zhang Y, Zhang H and Sun Z. 2003. Susceptibility of Mycobacterium

tuberculosis to weak acids. J Antimicrob Chemother, 52: 56-60.

Zink A, Haas CJ, Reischl U, Szeimies U and Nerlich AG. 2001. Molecular

analysis of skeletal tuberculosis in an ancient Egyptian population. J Med

Microbiol, 50 :355-366.

الملخص العربي

1


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


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


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 (


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


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

Education

Diagnosis of Mycobacterium Tuberculosis