tuberculosis. pathogenesis and lab diagnosis

84
Kingdom Of Saudi Arabia King Saud University College of Applied Medical Sciences Clinical Laboratories Sciences Department Tuberculosis (Mycobacterium Tuberculosis) Written By: Abdullah Said Al-Barakat U.N.: 424101197 Supervision: Dr. Jamal Eid Al-Said 1 st semester 1428/1429 H – 2007/2008 DH

Upload: bikoo555

Post on 10-Apr-2015

2.382 views

Category:

Documents


0 download

DESCRIPTION

This is my independent study, i did it in my college -Apllied Medical Sciences, Laboratories Department-

TRANSCRIPT

Page 1: Tuberculosis. Pathogenesis and lab diagnosis

Kingdom Of Saudi Arabia

King Saud University

College of Applied Medical Sciences

Clinical Laboratories Sciences Department

Tuberculosis

(Mycobacterium Tuberculosis)

Written By:

Abdullah Said Al-Barakat

U.N.:

424101197

Supervision:

Dr. Jamal Eid Al-Said

1st semester 1428/1429 H – 2007/2008 DH

Page 2: Tuberculosis. Pathogenesis and lab diagnosis

Contents

Introduction 5

Mycobacterium Tuberculosis

Morphology of Mycobacterium Tuberculosis 8

Virulence Factors of Mycobacterium Tuberculosis 11

1- Cord Factor(s) 11

2- Mycobacterial Sulfolipids 11

3- Mycoside 12

Transmission Of Mycobacterium Tuberculosis 13

Pathogenesis

Pathogenesis of Tuberculosis 15

1- Primary Tuberculosis 15

2- Post-primary Tuberculosis 18

3- Immunocompromised individuals 20

Symptoms Of Tuberculosis 21

Immunologic response

Immunologic Response Against Tuberculosis 23

Diagnosis Of Tuberculosis

Diagnosis Of Tuberculosis 30

1- Sputum Examination 30

1-1- Preparation, Staining and Microscopic

Examination

30

2- Culture Method 37

2-1- Preparation of Specimen 37

2-2- Incubation Conditions 37

2-3- Automated System 38

2-4- Identification 38

3- X-ray 39

4- Tuberculin Test 40

4-1- Description 40

4-2- Preparation 40

4-3- Aftercare 40

4-4- Risks 41

Page 3: Tuberculosis. Pathogenesis and lab diagnosis

4-5- Normal results 41

4-6- Abnormal Results 42

5- Polymerase Chain Reaction 43

New Diagnostic Method 46

1- Intended Used 46

2- Summary and Explanation of The Test 46

3- Principle of The Assay 47

4- Reagent and Storage 48

4-1- Peptide and Control Antigens 48

4-2- ELISA Component 48

4-3- Storage Instructions 48

5- Specimen Collection and Handling 49

6- Directions of Use 49

6-1- Stage One 49

6-1- Stage Two 51

7- Interpretation of Results 55

8- Warnings and Precaution 56

8-1- Warnings 56

8-3- Precaution 56

BCG Vaccine

BCG Vaccine of Tuberculosis 59

1- BCG Vaccine 59

2- Storing and Validity 59

3- Indication 59

4- Complication 60

5- BCG Vaccine and Other Vaccines 60

6- Administration and Dosage 60

7- Site 61

Treatment

Treatment of Tuberculosis 63

1- General Roles of Tuberculosis Treatment 63

2- Directly Observed Treatment, Short Course (DOTS) 63

3- Phases of Treatment 63

4- Hospitalization 64

Page 4: Tuberculosis. Pathogenesis and lab diagnosis

5- Duration of Treatment 64

6- General Procedure that should be followed during

Treatment

64

7- Categories of Treatment 65

8-1- CAT 1 65

8-2- CAT 2 65

8-3- CAT 3 66

8-4- CAT 4 66

67

Extrapulmonary Tuberculosis

1- Hepatic 69

1-1- Clinical 69

1-2- Diagnosis 70

2- Meninges 71

2-1- Clinical 71

2-2- Diagnosis 72

Conclusion 72

References 76

Figures References 83

Page 5: Tuberculosis. Pathogenesis and lab diagnosis

Introduction Tuberculosis (TB) is an infection caused by a germ called the tubercle

bacillus or Mycobacterium tuberculosis. Until effective anti-tuberculosis

drugs were introduced about 50 years ago, TB was one of the main causes

of death.

TB is still a major problem in many countries. It has been on the increase

in the developed world in recent years, probably because of increased air

travel and movement of people from areas where it is common. (97)

Tuberculosis most commonly attacks the lungs (as pulmonary TB) but

can also affect the central nervous system, the lymphatic system, the

circulatory system, the genitourinary system, bones, joints and even the

skin. Other mycobacteria such as Mycobacterium bovis, Mycobacterium

africanum, Mycobacterium canetti, and Mycobacterium microti can also

cause tuberculosis, but these species do not usually infect healthy adults.

Over one-third of the world's population has been exposed to the TB

bacterium, and new infections occur at a rate of one per second. (96)

A person may have had an infection with tuberculosis without being

aware. This can be discovered by a tuberculin skin test, the Heaf (or

Mantoux) test. When positive, it indicates that the person has a degree of

natural immunity. People who test negative do not have this immunity

and are more susceptible to infection by TB.

Tuberculin-negative people may benefit from BCG inoculation. This uses

a vaccine made from a modified version of the TB germ. It reduces the

risk of developing TB in about 70% of those vaccinated for

approximately 15 years. (97)

In 2004, mortality and morbidity statistics included 14.6 million chronic

active TB cases, 8.9 million new cases, and 1.6 million deaths, mostly in

developing countries. In addition, a rising number of people in the

developed world are contracting tuberculosis because their immune

systems are compromised by immunosuppressive drugs, substance abuse,

or HIV/AIDS.

Page 6: Tuberculosis. Pathogenesis and lab diagnosis

The rise in HIV infections and the neglect of TB control programs have

enabled a resurgence of tuberculosis. The emergence of drug-resistant

strains has also contributed to this new epidemic with, from 2000 to 2004,

20% of TB cases being resistant to standard treatments and 2% resistant

to second-line drugs. TB incidence varies widely, even in neighboring

countries, apparently because of differences in health care systems. The

World Health Organization declared TB a global health emergency in

1993, and the Stop TB Partnership developed a Global Plan to Stop

Tuberculosis aiming to save 14 million lives between 2006 and 2015. (96)

Page 7: Tuberculosis. Pathogenesis and lab diagnosis

������������

������ ����

Page 8: Tuberculosis. Pathogenesis and lab diagnosis

Morphology of Mycobacterium tuberculosis

The mycobacteria are rode – shaped that do not form spores. Although

they do not stain readily, once stained they resist decolorization by acid

or alcohol and are therefore called "acid-fast" bacilli.

Figure 1: Mycobacterium tuberculosis

In tissue, tubercle bacilli are thin straight rods measuring 0.4×3 um.

On artificial media, coccid and filamentous forms are seen with variable

morphology from one species to another. Mycobacteria can not be

classified as either gram-positive or gram-negative. Once stained by basic

dyes they can not be decolorized by alcohol, regardless of treatment with

iodine.

Figure 2: Mycobacterium tuberculosis - Ziehl Neelsen stain

Page 9: Tuberculosis. Pathogenesis and lab diagnosis

Mycobacteria are obligatory aerobes and derive energy from the

oxidation of many simple carbon compounds. Increased CO2 tension

enhances growth. The growth rate is much slower than that of most

bacteria. The doubling time of tubercle bacilli is about 18 hours.

Saprophytic forms tend to grow more rapidly, to proliferate well at 22-33

C, to produce more pigment, and to be less acid-fast than pathogenic

forms.

Mycobacteria are rich in lipids. These include mycolic acids, complex

waxes, and phospholipids. In the cell, the lipids are largely bound to

proteins and polysaccharides. (1)

Figure 3: Complex cell wall structure of Mycobacteria

The mycolic acids containing extremely long (C78-C90) side chains are

joined to the muramic acid moiety of the peptidoglycan by

phosphodiester bridges and to arabinogalactan by esterified glycolipid

linkages. Species variations are characterized by variation in sugar

substitution in the glycolipids or peptidoglycolipids. The mycobacterial

Page 10: Tuberculosis. Pathogenesis and lab diagnosis

cell wall is acid fast. This important property allows differential staining

in contaminated clinical specimens such as sputum.

This unusual cell wall structure endows mycobacteria with resistance

to dehydration, acids, and alkalis. The resistance to acids and alkalis is

useful in the isolation of mycobacteria from contaminated clinical

specimens such as sputum.

Another important consequence of the unique cell wall structure of

mycobacteria is the adjuvant action of whole cells when mixed with a

wetting agent in an oil-water emulsion. Such a mixture is called Freund's

complete adjuvant.

Although mycobacteria are normally cultured from clinical material

by inoculation on to enriched agar media containing bovine serum

albumin, they can grow on a chemically defined medium containing

asparagine, glycerol, and micronutrients. Even under ideal culture

conditions M tuberculosis grow very slowly, with doubling times on the

order of 18 to 24 hours. This extremely slow growth, even in vivo, has

two consequences of clinical significance: 1- the infection is an insidious,

chronic process, with may take clinically patent, and 2- cultures

inoculated with clinical material may take 4 to 6 weeks to exhibit

identifiable mycobacterial colonies. (2)

Page 11: Tuberculosis. Pathogenesis and lab diagnosis

��

Virulence factors of Mycobacterium tuberculosis There is three major virulence factors from the outer layer of the

complex mycobacterial cell wall have been characterized molecularly:

MTB cord factor, Mycobacterial sulfolipids (SL), and mycoside.

1- Cord factor(s)

The Cord factor(s) are trehalose-6, 6'-dimycolates. (3)

When cord factor coated on to bacillus subtilis, inhibit the migration

of blood leukocyte and result in death of the mice when injected

intrapertioneally. (4)

The toxic effects of cord factor have been attributed to an interaction

with mitochondrial membranes resulting in reduction of the activity of

DNA-dependent microsomal enzymes in various tissues (lung, liver, and

spleen). (5)

The problem with ascribing cord factor with a major role in virulence

is its occurrence in nonpathogenic as well as pathogenic species of

mycobacteria. (6)

2- Mycobacterial sulfolipids (SL)

The SL is trehalose 2'-sulfates acylated with pthioceranic,

hydroxypthioceranic, or saturated straight-chain fatty acids. (7)

SLs kill mice when injected intrapertioneally and enhance the toxicity

of cord factor. (8)

The production of SLs by MTB correlates with their virulence; a

virulent strain is deficient and virulent strains produce SL abundantly. (9)

The SLs inhibit the fusion of MTB phagosome and lysosome, thus

allowing MTB to evade host microbcidal molecules. (10)

The SLs although inhibition of phagosome-lysosome fusion also may

be mediated by other molecules, such as ammonia produced by MTB. 7

Page 12: Tuberculosis. Pathogenesis and lab diagnosis

��

3- Mycoside:

The mycoside are specific-specific glycoloipids and peptidoglycolipid

of mycobacteria. (11)

The surface glycolipids of MTB consist of trehalose-containing

lipooligosaccharide. Biochemical differences between surface mycosides

of virulent MTB and nonpathogenic strains of MTB have been described. (12)

The certain mycosides of mycobacteria induce formation of an

electron transparent zone in bacilli phagocytized by macrophages. (13)

The role of the electron-transparent zone in protecting MTB against

intracellular killing has not been determined.

Discovered recently, an abundant lipoglycan of the mycobacterial cell

wall, lipoarabinomannan (LAM), has been ascribed virulence function(s). (12)

The LAM inactivates phagocytic cell, inhibits induction of cellular

genes, and counteracts macrophage activation. By modulating the

cytokine milieu toward one of deactivation, LAM may allow the

persistence of MTB within tissue. (9)

Other factor Sigma factors, which are small transcription factors, regulate the

transcriptional activity of MTB during its adaptive states, and may be

indispensable for its virulence. (14)

Page 13: Tuberculosis. Pathogenesis and lab diagnosis

��

Transmission of Mycobacterium tuberculosis When people suffering from active pulmonary TB coughs, sneeze,

speak, kiss, or spit, they expel infectious aerosol droplets 0.5 to 5 um in

diameter. A single sneeze, for instance, can release up to 40,000 droplets. (15)

People with prolonged, frequent, or intense contact are at highest risk

of becoming infected, with an estimated 22% infection rate. A person

with active but untreated tuberculosis can infect 10-15 other people per

year. (16)

There is other people there at risk, include people in areas where TB is

common, resident of high-risk congregate setting, patient

immunocompromised by condition such as HIV/AIDS, people who take

immunosuppressant drugs, and health care workers serving these high-

risk clients. (17)

Figure 4: Transmission of Mycobacterium tuberculosis

The Transmission can only occur from people with active-not latent-TB.

The probability of transmission from one to another depends upon the

number of infectious droplets expelled by a carrier, the effectiveness of

ventilation, the duration of exposure, and the virulence of the

M.tuberculosis strain. (18)

Page 14: Tuberculosis. Pathogenesis and lab diagnosis

��

������������

Page 15: Tuberculosis. Pathogenesis and lab diagnosis

��

Pathogenesis of Tuberculosis The tubercle bacillus owes its virulence to its ability to survive within

the macrophage rather than to the production of a toxic substance. The

mechanism of virulence is poorly understood and is almost certainly

multifactorial. The immune response to the bacillus is of the cell-

mediated type, which, if mediated by Th1 T helper cells, leads to

protective immunity, but the presence of Th2 cells facilitates tissue-

destroying hypersensitivity reactions and progression of the disease

process. The nature of the immune responses following infection change

with time so that human tuberculosis is divisible into primary and post-

primary forms with quite different pathological features.

1- Primary Tuberculosis

Figure 5: Pathogenesis TB infection

The site of the initial infection is usually the lung. Following the

inhalation of bacilli. These bacilli are engulfed by alveolar macrophage in

which they replicate to form the initial lesion or Ghon focus. Some bacilli

are carried in phagocytic cells to the hilar lymph nodes where additional

foci of infection develop. The Ghon focus, together with the enlarged

hilar lymph nodes, form the primary complex. In addition, bacilli are

seeded by further lymphatic and haematogenous dissemination in many

organs and tissue, including other parts of the lung. When the

bacilli enter the mouth, as when drinking milk containing M. bovis, the

primary complexes involve the tonsil and cervical nodes or the intestine,

often the ileocaecal region, and the mesenteric lymph nodes. Likewise,

the primary focus may be in skin, with involvement of the regional lymph

nodes. This form of tuberculosis was an occupational disease of

anatomists and pathologists and was termed prosector's wart.

Page 16: Tuberculosis. Pathogenesis and lab diagnosis

��

Within about 10 days of infection, clones of antigen-specific T

lymphocyte are produced. These release cytokines, notably interferon-�,

which activate macrophage and cause them to form a compact cluster,

and cause them to form a compact cluster, or granuloma, around the foci

of infection. These activated macrophages are termed epithelioid cells.

Some of them fuse to form multinucleate giant cells. The center of the

granuloma contains a mixture of necrotic tissue and dead macrophages,

which, form its cheese-like appearance and consistency, is referred to as

caseation.

Activated human macrophages inhibit the replication of the tubercle

bacilli, but there is no clear evidence that they can actually kill them.

Being metabolically very active, the macrophages in the granuloma

consume oxygen, and the resulting anoxia and acidosis in the center of

the lesion probably kills most of the bacilli. Granuloma formation is

usually sufficient to limit the primary infection: the lesions become

quiescent and surrounding fibroblasts produce dense scar tissue, which

may become calcified. Not all bacilli are destroyed: some remain in a

poorly understood dormant form which, when reactivated, causes post-

primary disease. Programmed cell death (apoptosis) of bacteria-laden

cells also by cytotoxic T cells and natural killer (NK) cells may also

contribute to protective immunity.

In a minority of cases one of the infective foci progresses and gives

rise to the serious manifestations of primary disease, including

progressive primary lesions, meningitis, pleurisy and other bones and

joints. If a focus ruptures into a blood vessel, bacilli are disseminated

throughout the body with the formation of numerous granulomata. This,

from the millet seed-like appearance of the lesions, is known as miliary

tuberculosis.

Page 17: Tuberculosis. Pathogenesis and lab diagnosis

��

2- Post-primary tuberculosis In many individuals, the primary complex resolves and the only

evidence of infection is a conversion to tuberculosis reactivity. After an

interval of months, years or decades, reactivation of dormant foci of

tubercle bacilli or exogenous re-infection may lead to post-primary

tuberculosis, which differs in several respects from primary disease.

Main differences between primary and post-primary

tuberculosis in non-compromised patients

Characteristic Primary Post-primary

Local lesion Small Large

Lymphatic

involvement

Yes Minimal

Cavity

formation

Rare Frequent

Tuberculin

reactivity

Negative (initially) Positive

Infectivity Uncommon Usual

Site Any part of lung Apical region

Local spread Uncommon Frequent

Endogenous reactivation may occur spontaneously or after an

intercurrent illness or other condition that lowers the host's immune

responsiveness. For unknown reasons reactivation or re-infection

tuberculosis often occurs in the upper lobes of the lungs. The same

process of granuloma formation occurs, but the necrotic element of the

reaction causes tissue destruction and the formation of large areas of

caseation termed tuberculomas. Proteases librated by activated

macrophages cause softening and liquefaction of the caseous material,

and an excess of tumor necrosis factor and other immunological

mediators cause the wasting and fevers characteristic of the disease.

Page 18: Tuberculosis. Pathogenesis and lab diagnosis

��

Figure 6: Infected lung show tuberculoma

The interior of the tuberculoma is acidic and anoxic and contains few

viable tubercle bacilli. Eventually, however, the expending lesion erodes

through the wall of a bronchus, the liquefied contents are discharged and

a well-aerated activity is formed. The atmosphere of the lung, with a high

carbon dioxide level, is ideal for supporting the growth of the bacilli, and

huge numbers of these are found in the cavity walls. For this reason,

closure of the cavities by collapsing the lung, either by artificial

pneumothorax or by excising large portions of the chest wall, was a

standard treatment for tuberculosis in the pre-chemotherapy area.

Page 19: Tuberculosis. Pathogenesis and lab diagnosis

Figure 7: Infected lung show the cavities

Once the cavity is formed, large numbers of bacilli gain access to the

sputum, and the patient becomes an open or infectious case. This is a

good example of the transmissibility of and a pathogen being dependent

upon the host's immune response to infection. Surprisingly, about 20% of

cases of open cavitating tuberculosis resolve without treatment.

In post-primary tuberculosis, dissemination of bacilli to lymph nodes

and other organs is unusual. Instead, spread of infection occurs through

the bronchial tree so that secondary lesion develops in the lower lobes of

the lung. Likewise, secondary lesions may occur in the trachea, larynx

and mouth, and swallowed bacilli cause intestinal lesions; secondary

lesions may also develop in the bladder and epididymis in cases of renal

tuberculosis. Post-primary cutaneous tuberculosis (lupus vulgaris) usually

affects the face and neck. Untreated, it is a chronic condition leading to

gross scarring and deformity. Some cases are secondary to sinus

formation between tuberculosis lymph nodes and the skin

(scrofuloderma).

Page 20: Tuberculosis. Pathogenesis and lab diagnosis

3- Immunocompromised individuals Reactivity tuberculosis is particularly likely to occur in

immunocompromised individuals, including the elderly and transplant

recipients; it often occurs early in the course of human immunodeficiency

virus (HIV) infection. Tuberculosis acts synergistically with HIV to lower

the patient's immunity and it is an AIDS-defining condition. As a result,

even if tuberculosis is treated effectively in HIV-positive patient, the

mortality rate due to other AIDS-related conditions is high, with many

dying within 2 years. Cavity formation is unusual in the more profoundly

immunocompromised patients, emphasizing the importance of the

immune response in this pathological process. Instead, diffuse infiltrates

develop in any part of lung. In contrast to post-primary disease in non-

immunocompromised individuals, lymphatic and hematogenous

dissemination are common. Sometimes there are numerous minute

lesions teeming with tubercle bacilli throughout the body – rapidly fatal

conditions termed cryptic disseminate tuberculosis. The interval between

infection and development of disease is considerably shortened in

immunocompromised persons. (19)

Page 21: Tuberculosis. Pathogenesis and lab diagnosis

��

Symptoms of Tuberculosis

When the disease becomes active, 75% of the cases are pulmonary

TB. Symptoms include chest pain, coughing up blood, and a productive,

prolonged cough for more than three weeks. Systemic symptoms include

fever, chills, night sweats, appetite loss, weight loss, pallor, and often to

tendency to fatigue very easily.

In the other 25% of active cases, the infection moves from the lung,

causing other types of TB more common in immunosuppresed persons

and young children. Extrapulmonary infection sites include pleura, the

central nervous system in meningitis, the lymphatic system in scrofula of

the neck, the genitourinary system is urogenital tuberculosis, and bones

and joints in Pott's disease of the spine. An especially serious form is

disseminated TB, more commonly Known as miliary tuberculosis.

Although extrapulmonary TB is not contagious, it may co-exist with

pulmonary TB, which is contagious. (96)

Page 22: Tuberculosis. Pathogenesis and lab diagnosis

��

����� �������������

Page 23: Tuberculosis. Pathogenesis and lab diagnosis

��

Immunologic Response against Tuberculosis Since Tb is basically a pulmonary disease, the lung is the point of

entry for the microorganism and the principle manifestation site of the

infection. Immediately after a primary infection, air particles, alveolar

macrophages, and dendritic cells, this phagocytosed the M. tuberculosis;

migrate through the lymphatic system toward the regional lymph node,

forming the Ghon complex. Simultaneously, phagocytic cell can

penetrate the pulmonary parenchyma, initiating an inflammatory focus to

which other macrophages will be attracted. In this case, microorganism

initiates the formation of a granuloma, coordinated by T lymphocytes.

The T cells granulomas, indispensable to the formation of stable

granulomas, contacting mononuclear phagocytes and influencing their

differentiation and activation status. The M. tuberculosis is contained in

the granuloma, and can persist in the lesions for decades, in latent form,

without triggering the disease.

The immunosuppression, either due to the poor health status of

individual, HIV infection, or use of immunosuppressant, is the most

frequent cause of the multiplication of bacilli enclosed in the granuloma

and of the reactivation of TB (endogenous reactivation), as compared to

the reinfection (exogenous) with M. tuberculosis. (20)

The macrophages in the tissue constitute one of the first lines of

defense against mycobacteria. After being phagocytosed, the bacilli

remain within the phagosome. After the phagosome-lysosome fusion,

antigens can be processed and subsequently presented to T- helper (TH)

lymphocytes (CD4+), through the major histocompatibility complex class

�� (MHC ��) molecules (also known as antigen-presenting cells), which

are found only in macrophages, dendritic cells, and B lymphocytes. It is

known that T- helper type 1 (Th1) CD4+ cells play the principle role in

the immune response to mycobacteria.

Also they said the cytotoxic T cells (CD8+), which recognize from the

cytoplasm (tumor or viral), also participate in the immune response to M.

tuberculosis. (21)

The CD8+ T cells can recognize peptide fragments bound to MHC

class � cells, which are expressed in practically all differentiated or

mature cells of the organism. In the case of mycobacteria, it has been

demonstrated that apoptotic vesicles from infected cells containing

Page 24: Tuberculosis. Pathogenesis and lab diagnosis

��

antigens of the bacillus with MHC class � can specifically stimulate

CD8+ T cells. (22)

In a phenomenon know as cross-presentation, antigens of intracellular

pathogens can directly access the presentation via MHC class � cells,

owing to the capacity of the phagosome to fuse with the endoplasmic

reticulum to the phagosome. Consequently, phagocytosed antigens can

access the cytoplasm, suffer degradation by proteases, known as

proteases, return to the phagosome through transporters associated with

antigen processing (TAPs), and bind to MHC class � molecules located in

the phagosome, leading to the subsequent expression on the cell surface

and to the recognition by CD8+ cells. (23)

Atypically lymphocytes (CD4- and CD8-) have receptors containing

gamma/delta polypeptide chains and recognize phosphoric components of

M. tuberculosis. (24)

The regardless of MHC class � or ��, whereas T lymphocyte receptors

restricted only to CD1 can be stimulated by glycolipids derived from the

cell wall of the mycobacteria. (25)

The immune system can recognize and effectively respond to a broad

spectrum of antigenic determinants of different biochemical

characteristics. In this recognition, there is a hierarchy among the T cell

subpopulations that contribute to the immune response to mycobacteria,

and the CD4+ and CD8+ T lymphocytes are the most important in this

hierarchy. (26)

Regarding the innate immune response, neutrophils are the first

inflammatory cells to arrive at the bacillus multiplication site, followed

by natural killer (NK) cells and macrophages. The NK cells can destroy

pathogens directly or by killing the infected monocytes, as well as being

able to activate phagocytic cells at the site of infection. (21)

However, it has been shown that mice depleted of NK1.1 cells do not

present greater susceptibility to mycobacterial infection. (27)

The recognition and phagocytosis of bacteria by innate immunity cells

(neutrophils, macrophages, and dendritic cells) occur via recognition

receptors, such as the mannose receptor, receptors for the Fc portion of

antibodies (FcRs), and receptors for activation products of the

complement system, such as C3b and C4b (CR1), among others. (21)

Page 25: Tuberculosis. Pathogenesis and lab diagnosis

��

The activation of standard recognition receptors, such as toll-like

receptors (TLRs), leads to an important connection between innate and

acquired immune response. The expression of co-stimulating molecules

such as CD80 and CD86, on the surface of macrophages and dendritic

cells, is induced after TLRs recognize specific molecules of the

pathogens, such as lipoarabinomannans, lipoproteins and other lipid

derivatives of M. tuberculosis. (28)

The activation of CD4+ lymphocytes involves the recognition of the

peptide bound to MHC class �� and the interaction between co-stimulating

molecules, such as interleukin (IL)-12, and cytokines produced by

activated T lymphocytes, such as IL-2, are involved in the activation and

proliferation of T lymphocytes. Consequently, M. tuberculosis-specific

antigens interact with TLRs and other receptors present on the surface of

macrophages and dendritic cells, thereby inducing a predominantly pro-

inflammatory cellular immune response.

Figure 8: Mechanisms involved in the activation of macrophages

and T lymphocytes by mycobacteria

Page 26: Tuberculosis. Pathogenesis and lab diagnosis

��

Cytokines, molecules produced and secreted by different

immunocompetent cells after some stimulus, are a central component in

the defense against mycobacteria. At all stages of the immune response,

the cytokines produced participate in the regulatory processes, as well as

in effector functions. (21)

The recognition of the mycobacteria and posterior secretion of IL-12 by

macrophages are processes initiated before the M. tuberculosis antigens

are presented to T lymphocytes. The production of interferon-gamma

(INF-�) in NK cells is induced by Il-12 in the initial phase of the immune

response. In addition, IL-12 induces the activation, differentiation, and

production of INF-�, as well as the expansion of antigen-specific TH1

cells. Recently, other cytokines have been described, produced by

macrophages and dendritic cells, which present similar activity to that of

IL-12. The production of INF-� is also induced by IL-23, IL-18, and IL-

27, a process that is accelerated when IL-18 and IL-27 act in synergy with

IL-12. It is believed that IL-27 acts in an early phase of the immune

response, preceding IL-12 in the inducement of the production of INF-�,

whereas IL-12 present strong activity in the amplification of INF-�

production and Th1 lymphocyte expansion at a subsequent stage. (29)

Constituting the principle immune response, Th1 cells are necessary for

the control of the chronic phase of the infection, due to the effect that IL-

2 and IFN-� have on T cells and macrophages. Produced by dendritic

cells and macrophages, IL-12 is active in T cells, forming a link between

the innate and acquired responses. Individuals with mutations in genes

IL-12p40 and IL-12R present reduced T-cell production of IFN-� and are

more susceptible to infections disseminated by the bacilli Calmette-

Guerin (BCG) vaccine and M. avium. (30)

The bacterial capacity of the macrophage against M. tuberculosis

needs to be previously activated, and IFN-� is the principle and most

potent mediator of this process. (31)

Increased production of IFN-� can have a variety of effects: increasing

the expression of various genes in the macrophages; increasing the

expression of the MHC (greater presentation of antigens) and of

immunoglobulin receptors (FcRs; greater capacity for phagocytosis);

recruiting T lymphocytes that participate in the destruction of bacteria;

and promoting the production of nitric oxide. Although IFN-� production

alone cannot control the bacillus, IFN-� is one of the crucial components

of the protective response against the pathogen. (21, 30, 31)

Page 27: Tuberculosis. Pathogenesis and lab diagnosis

��

In synergy with tumor necrosis factor alpha (TNF- ), IFN-� genes or

IFN-� receptors predispose individuals to serious mycobacterial

infections. (32)

Although the production capacity of IFN-� can vary among individuals,

some studies suggest that IFN-� levels are decreased in patients with

active TB. (33)

These levels are even lower in patients with advanced pulmonary disease. (34)

In addition, it has been demonstrated that M. tuberculosis can prevent

macrophages from adequately responding to IFN-�. (35)

However, the importance of IFN-� in the protection against various

pathogens, including parasites, bacteria, and viruses, has been well

established. (36)

In various biological systems, is frequently used as a marker of effector

cell activity. Cytokines such as IL-4, IL-5, andIL-10, which are involved

in the activation of B cells and the production of antibodies, are produced

by Th2 cells. However, immunity against Tb is mediated by Th1 cells.

Nevertheless, it has been recently reported that, in human TB, in addition

to the Th1-produced cytokines, IL-4 is produced. (37)

It has been demonstrated that, due to the strong antagonist effect that IL-4

has on the Th1 response, that response can be jeopardized even when the

Th2 response is weak. (38)

The TLR2 expression and the activation of macrophages can be

negatively regulated by IL-4. (28)

Recently, CD4+ and CD25+ regulatory T cells have been identified.

These cells produce IL-10 and transforming growth factor-bate, as well as

expressing TLRs (which can react with mycobacteria) and participating

in the suppression of protective immunity. Therefore, they constitute a

potentially important factor at the latency or progression of TB. (20)

The immune system contains molecule known as chemokines, which

induce chemotaxis or signaling. Chemokines can potentially intensify the

immune response through their capacity to recruit and focus distinct

populations of leukocytes. In in vivo and in vitro murine models, M.

tuberculosis induces the production of a variety of chemokines, including

macrophage inflammatory protein 1-alpha (MIP-1 ), MIP-2, monocyte

Page 28: Tuberculosis. Pathogenesis and lab diagnosis

��

chemoattractant protein 1 (MCP-1), MCP-3, MCP-5, and IFN-�-inducible

protein 10. (39)

The production of IFN-� can regulate that of various chemokines. The

monokine induced by IFN-� (MIG, or CXCL9) can accomplish this task

and be used as a sensitive and specific measure of IFN-� production. One

of the primary effects of IFN-� release is macrophage production of MIG. (40) It is believed that MIG is an important mediator of the protective immune

response. In fact, peripheral blood mononuclear cells of patients with TB

produce MIG in response to M. tuberculosis-specific antigens, and this

production is significantly lower in control individuals residing in an

endemic area and vaccination with BCG. (41)

Page 29: Tuberculosis. Pathogenesis and lab diagnosis

�����������

������ ����

Page 30: Tuberculosis. Pathogenesis and lab diagnosis

Diagnosis of tuberculosis

Method of diagnosis:

1- Sputum examination:

The main method for diagnosis of tuberculosis is smear examination.

Direct sputum examination of suspects should be performed and three

sputum samples should be collected during a period of two days. The first

sample has to be collected in the first interview with the patient (On spot)

the second sample should be a morning sample collected by the patient at

home and the third sample should be collected on the second interview

(on spot). The patient should collect the sample in a good ventilated room

away from other patients under supervision of trained personal. The

patient mouth should be cleaned from food remnants. If the first sample

was found to be positive and the patient did not come for the second

sample, he should be traced again to complete the samples. During

waiting for smear results, nonspecific broad spectrum antibiotics and

symptomatic medications could be administered, if required. If there is no

improvement by this medication and smear result were found to be

negative, the patient must be examined clinically and by X-ray with

collection of another set of sputum samples.

At the end of treatment, there may be difficulty in collecting sputum

sample but a sample should be collected and if the laboratory results

denote that the sample is saliva, the result is considered as smear

negative.

1-1 Preparation, staining and microscopic examination:

1-1-1 Sputum collection:

General rules:

* A trained person should supervise collection of the sample, as the

sample collected under supervision is better than that collected without.

* The sample must be collected either out side in the open air or in a good

ventilated room specified for this purpose.

* The sample should be collected away form other patients.

Page 31: Tuberculosis. Pathogenesis and lab diagnosis

��

Preparation for sputum sample collection:

a- The patient's mouth should be cleaned from food remnants by washing

with water.

b- Fill-in the sputum examination request from.

c- Explain to the patient reason for the examination, benefits of the

examination and how to cough so that the expectoration will come from

as deep down in the chest as possible.

Technique for collection:

a- Ask the patient to cough deeply.

b- Be sure that no one standing in front the patient mouth to prevent

contamination of the outer sides of it. If this occurred recollect another

sample using a clean container and dispose the contaminated one.

Procedures after collection:

a- Close securely the sputum container.

b- Wash your hand with a disinfectant.

c- Keep the sample in as cool as possible place, refrigerator or a cooler

box.

d- Preferably, appointments for sputum collection should be given before

the date of transportation by 24 hours.

e- Give the patient a new container and make quite sure that he has

understood that he must spit into the container as soon as he coughs up

sputum in morning.

Transport of sputum specimens:

Sputum specimens should be transported to the laboratory as soon as

possible (within seven days from collection) and it should be stored in as

cool place as possible. Samples for each patient should be accompanied

with a sputum examination request form.

Page 32: Tuberculosis. Pathogenesis and lab diagnosis

��

1-1-2 Sputum examination:

Microscopy:

a- Sputum samples should be examined by direct microscopy using light

microscope and Ziehl Nelseen Stain. A trained laboratory specialist or a

technician should examine the sputum.

b- Fluorescence microscope using auramine stain could be used. It is fast

but the rate of false positive results is some what high. Fluorescence

microscopes are only available in the main laboratories.

1-1-3 Preparation of smears:

Staining looks for the ability of mycobacteria to resist decolonization

on exposure to acidic alcohol. This could be evidenced by the presence of

red colored bacilli among a blue background or the presence of illuminant

bacilli in case of auramine.

The smear is prepared as follow:

* Using a loop, pick a small portion of sputum selecting purulent

particles if present, and put it on an engraved clean slid with the number

of specimen. The slide must be cleaned with alcohol before use.

* Spread the sputum samples as thinly as possible over two thirds of

the slid.

* Sterilize the loop between successive specimens by holding in the

flam until the wire is red hot.

* Fix the sample by passing it three times through the flam. The

smear must be uppermost and not facing the flam (never overheat the

slide).

1-1-4 Staining:

1-1-4-a: Ziehl Nelseen Stain

a- Place the slides on the slid rack with the smeared sides uppermost,

their edges separated and the numbers turned toward the operator. The

smeared part of each slide can be covered with a piece of filter paper.

b- Cover the whole surface of the slides with Ziehl's carbol fuchsin.

Page 33: Tuberculosis. Pathogenesis and lab diagnosis

��

c- Heat very gently until vapor rises, use the flam of Bunsen-burner or of

a wad of cotton-wool in alcohol fixed on the end of a metal rod or a fairy

strong stick of wood.

In no case must the stain boil or dry on the slide. If the stain

accidentally runs away, add more and heat again. Leave the warm stain

for five minutes.

Decolorization:

a- With the forceps, remove the filter paper and deposit them in the waste

receptacle.

b- Rinse each slide individually in a gentle stream of running water (tap

water or bottled water) until all free stain is washed away.

c- Replace all slides on the slide-rack and cover each one individually

with 25% sulphuric acid for three minutes.

d- Rinse as in (b) above.

e- Decolorize again for 1-3 minutes as in (c) above until all color has

practically disappeared.

f- Rinse as in (b) above.

Counter-staining:

a- Replace decolorized, rinsed slides on slide-rack and flood smear with

0.3% methylene blue counter-stain for 60 minutes.

b- Rinse as in (1-1-5-b) above and allow to dry in open air.

Examination by microscopy:

For the examination of stained specimens, a binocular microscope is

most convenient, with an immersion objective (X100) and eye piece of

moderate magnification (X6 or X8). Nevertheless, if there is no

electricity, and in hot or humid conditions, a monocular microscope

might be better, because there are fewer surfaces to be attacked by fungi.

If no electricity is available, day light must used as light source and

the table with the microscope must be placed immediately before a

window.

Page 34: Tuberculosis. Pathogenesis and lab diagnosis

��

Use of the microscope:

Before starting the actual examination of smears, the technician must

make sure that all elements of this microscope are correctly set. He

should, in particular, check that the source of light is well regulated and

focused, that the condenser is in the upper position, with the diaphragm

open and that the immersion objective and the ocular are clean.

Put a drop of immersion oil on the left edge of the stained smear (near

the engraved number) and place the slide on the microscope stage. To

avoid possible contamination of the immersion oil, do not touch the slide

with oil applicator, but permit the drop of oil to fall freely onto the slide.

With the macrometric screw, lower the immersion lens, keeping

continuous watch until it touches the drop of oil. Looking through the eye

pieces bring the immersion lens slowly upwards, by means of the

micrometric screw. All during the reading, the correct focusing is ensured

by using the micrometric screw.

Technique of reading:

Examine at least 100 microscopic fields. For a skilled microscopist,

this will take 5 minutes.

The reading must be systemic and standardized. For instance, begin

the reading of the slide in the center of the left end of the smear, by light

adjustment of the micrometric screw, systemically examine the fields,

beginning at the periphery and ending at the center.

After examining a microscopic field, move the slide longitudinally so

that the neighboring to the right can be examined. In this smear, all the

microscopic fields from beginning to end of this central length of the

slide should be examined.

The number of microscopic fields in one length of the slide

corresponds to at least 100.

When no acid-fast bacilli (AFB) are found in 100 fields, a more

through fields should be made in 100 new fields.

Tubercle bacilli look like red rods, slightly curved, more or less

granular, isolated, in pairs or in groups, standing out clearly against the

blue background. Count the number of AFB and report this number in the

notebook.

Page 35: Tuberculosis. Pathogenesis and lab diagnosis

��

Figure 9: Mycobacterium tuberculosis - Ziehl Nelseen Stain

At the end of examination, take the slide from the microscope stage,

check the identification number engraved on it, and enter the result of the

examination in the lat column of the dispatch list. Dip the slide into

toluene (or xylol) to remove the immersion oil and place it in box foe

examined slides.

Before examining the next slide, wipe the immersion lens with a piece

of clean cotton. (42)

1-1-4-b: Auramine stain

a- Place the slides on the slid rack with the smeared sides uppermost,

their edges separated and the numbers turned toward the operator. The

smeared part of each slide can be covered with a piece of filter paper.

b- Heat-fix dried smear.

c- Cover the fixed smear with the auramine-phenol stain for 10 minutes.

d- Wash off stain with clean water.

Page 36: Tuberculosis. Pathogenesis and lab diagnosis

��

e- Decolorize the smear by covering it with 1% acid alcohol for 5

minutes.

f- Wash off the acid alcohol with clean water.

g- Cover the smear with the potassium permanganate solution for about

10 seconds, followed by several rinses with clean water.

d- Wipe the back of the slide clean and place it in a draining rack for the

smear to dry. Do not blot dry. To prevent fading of the fluorescence,

protect the stained smear from sunlight and bright light.

e- Systematically examine the smear for AFB by fluorescence

microscopy using 40X objective.

Result:

Acid fast bacilli (AFB)….White-yellow rods glowing against dark

background.

Figure 10: Fluorescence microscope

Page 37: Tuberculosis. Pathogenesis and lab diagnosis

��

Figure 11: Mycobacterium tuberculosis - Auramine Stain

Reporting sputum smears:

When fluorescence AFB are seen, report the smear as 'AFB

positive', and give an indication of the number of bacilli present

in plus signs (+ to +++). (98)

2- Culture method

As sputum and certain other specimens frequently contain many

bacteria and fungi that would rapidly overgrow any mycobacteria on the

culture media, these must be destroyed. Decontamination methods make

use of the relatively high resistance of mycobacteria to acids, alkalis and

certain disinfectants.

2-1 preparation of sputum:

In the widely used Petroff method, sputum is mixed well with 4%

sodium hydroxide for 15-30 minutes, neutralized with potassium

dihydrogen orthophosphate and centrifuged. The deposit is used to

inoculate LJ or similar media.

2-2 Incubation condition:

Inoculated media are incubated at 35-37 C and inspected weekly for at

least 8 weeks. Any bacteria growth is stained by the ZN method and, if

acid-fast, it is subcultured for further identification.

Page 38: Tuberculosis. Pathogenesis and lab diagnosis

��

Figure 12: Mycobacterium tuberculosis LJ media

2-3 Automated system:

A more rapid bacteriological diagnosis is achievable by use of

commercially available automated systems. Systems that detect color

changes in dyes induced by the release of carbon dioxide, or the

unquenching of fluorescent dyes on the consumption of oxygen by

metabolizing bacilli, have replaced the earlier radiometric method.

2-4 Identification:

The first step in identification is to determine whether an isolate is a

member of the M. tuberculosis complex. These organisms:

* Grow slowly.

* Do not produce yellow pigment.

* Fail to grow at 25 and 41 C.

* Do not grow on egg media containing p-nitrobenzoic acid (500mg/l). (19)

Page 39: Tuberculosis. Pathogenesis and lab diagnosis

3- X-ray

Diagnosis by means of radiological examination in patients suspected

of tuberculosis is unreliable. Abnormalities identified on a chest

radiograph may be due to tuberculosis or to a variety of other conditions,

and the appearance on the radiograph is not specific for tuberculosis. So,

it is recommended to diagnose tuberculosis by direct smear examination

for acid fast bacilli. Chest radiograph may be helpful in those patients

who are not sputum smear positive, for assessment of contacts and in

diagnosis of military tuberculosis with smear negative results, but it

should be read by a competent physician. (42)

Figure 13: X-ray show Cavitations in lungs

Page 40: Tuberculosis. Pathogenesis and lab diagnosis

4- Tuberculin testing

4-1 Description TB skin tests are usually given at a clinic, hospital, or doctor's office.

Some times the tests are given at schools or workplaces and may be a pre-

employment requirement. Many cities provide free TB skin tests and

follow up care. The Mantoux PPD tuberculin skin test involves injecting

a very small amount of a substance called PPD tuberculin just under the

top layer of the skin (intracutaneously). Tuberculin is a mixture of

antigens obtained from the culture of M. tuberculosis. Antigens are

foreign particles or proteins that stimulate the immune system to produce

antibodies. The latter of PPD it is mean Purified Protein Derivative. The

latter is the preferred testing substance. The test is usually given on the

inside of the forearm about halfway between the wrist and the elbow,

where a small bubble will form as the tuberculin is injected. The skin test

takes just a minute to administer.

After 48-72 hours, a trained person for evidence of swelling will

examine the test site. People who have been exposed to tuberculosis will

develop an immune response, causing a slight swelling at the injection

site. If there is a lump or swelling, the health care provider will use a ruler

to measure the size of the reaction. Some public health physicians

recommend using a 72-hour waiting period as a general practice on the

grounds that a 48-hour waiting period yields a higher percentage of false

negative test results.

4-2 Preparation

There is no special preparation needed before a TB skin test. A brief

personal history will be taken to determine whether the person has had

tuberculosis or a TB test before, has been in close contact with anyone

with TB, or has any significant risk factors. Directly before the test, the

skin on the arm at the injection site is usually cleaned with an alcohol

swab and allowed to air dry.

4-3 Aftercare

After having a TB skin test, it is extremely important to make sure that

the patient keeps the appointment to have the test reaction read. The

patient is instructed to keep the test site clean, uncovered, and to not

scratch or rub the area. Should severe swelling, itching, or pain occur, or

Page 41: Tuberculosis. Pathogenesis and lab diagnosis

��

if the patient has trouble breathing, the clinic or health care provider

should be contacted immediately.

4-4 Risks

The risk of an adverse reaction is very low. Occasionally, an

individual who has been exposed to the TB bacteria will develop a large

reaction in which the arm swells and is uncomfortable. This reaction

should disappear in two weeks. A sore might develop where the injection

was given, or a fever could occur, but these are extremely rare reactions.

It is possible that a person who has TB will receive a negative test

result (called a "false negative") or a person who does not have TB may

receive a positive test result (called a "false positive"). If there is some

doubt, the test may be repeated or the person may be given a diagnostic

test using a chest x ray and/or sputum sample culture test to determine

whether the disease is present and/or active in the lungs.

Figure 14: Swelling skin (Tuberculin test)

4-5 Normal results

In people who have not been exposed to TB, there will be little or no

swelling at the test site after 48-72 hours. This is a negative test result.

Negative test results can be interpreted to mean that the person has not

been infected with the tuberculosis bacteria or that the person has been

Page 42: Tuberculosis. Pathogenesis and lab diagnosis

��

infected recently and not enough time has elapsed for the body to react to

the skin test. Persons become sensitive between two and ten weeks after

the initial infection. As a result, if the person has been in contact with

someone with tuberculosis, the test should be repeated in three months.

Also, because it may take longer than 72 hours for an elderly individual

to develop a reaction, it may be useful to repeat the TB skin test after one

week to adequately screen these individuals. Immunocompromised

persons may be unable to react sufficiently to the Mantoux test, and either

a chest X- ray or sputum sample may be required.

A newer test that appears to be preferable to the tuberculin skin test in

evaluating patients who are HIV-positive is the enzyme-linked

immunospot (ELISPOT) assay. A group of researchers in the United

Kingdom found that the ELISPOT assay was more accurate than the PPD

test in detecting active as well as latent tuberculosis in HIV-positive

patients.

4-6 Abnormal results

A reaction of 5 mm of induration (swelling) is considered positive for

the following groups:

• Household contacts of persons with active tuberculosis

• AIDS patients

• Persons with old healed tuberculosis on chest X-ray

• Organ transplant recipients.

• Persons receiving immunosuppressive medications

A reaction of 10 mm of induration is considered positive in individuals

with one or more of the following risk factors which are either reason to

have a higher exposure to TB and/or a condition that increases the risk for

progression to active TB:

• Foreign-born immigrants from Asia, Africa, or Latin America

• Injection drug users and persons who abuse alcohol

• Residents and employees of such high-risk congregate settings as

hospitals, homeless shelters, and jails

• Medically under-served low income populations

• TB laboratory personnel

• Children younger than four years of age or infants, children or

adolescents exposed to adults in high risk categories

• Residents of long-term care facilities

Page 43: Tuberculosis. Pathogenesis and lab diagnosis

��

• Individuals with certain medical conditions that increase the risk of

developing tuberculosis; these medical conditions include being

10% or more below ideal body weight, silicosis, chronic renal

failure, diabetes mellitus, high dose corticosteroid or other

immunosuppressive therapy, some blood disorders like leukemia

and lymphomas, and other cancer

Finally, a reaction of 15 mm of indurations or greater is considered

positive in those with no risk factors and are therefore at the lowest risk

of developing TB.

A TB skin conversion is defined as an increase of 10 mm or greater of

indurations within a two year period, regardless of age.

A positive reaction to tuberculin may be the result of a previous natural

infection with M. tuberculosis, infection with a variety of non-

tuberculosis mycobacteria (cross-reaction), or tuberculosis vaccination

with a live, but weakened (attenuated) mycobacterial strain. TB

vaccination is not done in the US. Cross-reactions are positive reactions

that occur as a result of a person's exposure to other non-tuberculosis

bacteria. These tend to be smaller than those caused by M. tuberculosis.

There is no reliable way of distinguishing whether a positive TB skin test

is due to a previous vaccination against tuberculosis. Generally, however,

positive results are not due to vaccination exposure because reactions in

vaccinated people tend to be less than 10 mm, and an individual's

sensitivity to tuberculin steadily declines after vaccination. If the skin test

is interpreted as positive, a chest x ray will be performed to determine

whether the person has active tuberculosis or whether the body has

sufficiently handled the infection. (43)

5- Polymerase Chain Reaction:

Since its introduction in 1985, the polymerase chain reaction (PCR)

has transformed the way DNA analysis is performed. (44) This process

involves the in vitro synthesis of millions of copies of a specific DNA

segment and is based on the annealing and extension of two

oligouncleotide primers that flank the target area in the DNA. First, the

DNA is denatured and then each primer hybridizes to one of the two

separated standards so that extension from each 3' hydroxyl end is

directed toward the other. The annealed primers are extended on the

template strand with a DNA polymerase. These three steps (denaturation,

primer binding, and DNA synthesis) represent a single PCR cycle.

Repeated cycles of denaturation, primer annealing, and extension produce

Page 44: Tuberculosis. Pathogenesis and lab diagnosis

��

an exponential accumulation of a discrete fragment (target). PCR can

amplify single or double-stranded DNA, and RNA can serve as a target if

reverse transcription is used to make a DNA copy. This technology

permits amplification of a highly specific DNA segment into millions or

billions of copies in only a few hours. Thus, when once it would have

been almost impossible to find a single DNA segment in a sample, PCR

permits the amplification of this DNA to such a quantity that it can be

detected by simple laboratory means. This technology has made possibly

new methods for the diagnosis of many infectious diseases, including

tuberculosis.

In order to use PCR for detection M. tuberculosis in clinical samples,

it first was necessary to identify and characterize a DNA segment within

the M. tuberculosis chromosome specific and unique for this organism.

Hance et al. reported the detection of mycobacteria by PCR using a

segment of DNA that codes for the 65-kDa antigen (the gro EL heat

shock protein) as the target. However this DNA segment is present in all

mycobacterial species and is not specific for M. tuberculosis. (45) Also,

PCR using this target technique has not been shown to be sensitive

enough for use in clinical samples and it is unlikely to be adopted for

widespread clinical use. Manjunath identified a target segment of DNA

specific for M. tuberculosis, and additional PCR methods for the

diagnosis of tuberculosis have been reported by Pao et al., by Shanker et

al., by Sjobring et al., and by Plikaytis et al. (46-50) Boddinghaus and

colleagues have used the 16S ribosomal gene as a PCR target, a segment

that is conserved in all microbial species. This target offers tha advantage

of a high copy number of rRNA sequences, but despite this apparent

advantage, the reported sensitivity is no higher than that obtained with

single-target DNA sequences. (51)

The most attractive target specific for M. tuberculosis and M. bovis is

that described by Eisenach et al. (52) The target sequence is repeated

within the M. tuberculosis chromosome up to 20 or more times and each

individual copy can be amplified using same primers. This duplication

increases the sensitivity by a factor up to 20 or more compared to those

methods that utilize a chromosomal target that occurs only once per

chromosome. The target sequence is part of a larger repeated segment

that most probably is an insertion sequence that has been designated

IS6110. (53)

In a clinical trial of 314 sputum samples, 93% of the patients with

tuberculosis were PCR positive. (54) Among the 104 PCR positive

patients, 83 were smear and culture positive, 2 were smear and culture

Page 45: Tuberculosis. Pathogenesis and lab diagnosis

��

negative. Four patients who had completed or partially completed

chemotherapy had PCR positive specimens. Of the 136 specimens

obtained from patients who did not have tuberculosis (72 had

nontuberculous mycobacterial infection and 64 had no known

mycobacterial infection), there were 4 specimens found to be PCR

positive. This study demonstrated the utility of the IS6110 PCR assay and

it is expected that this assay will be adapted for use to detect M.

tuberculosis in clinical samples of cerebrospinal fluid, blood, and tissue.

This test will detect low numbers of organisms in a sample, perhaps as

few as 10 under circumstances and it will detect nonviable organisms as

well.

Both RNA and DNA amplification systems are commercially

available. (55, 56) The PCR technique can be performed on sputum, spinal

fluid, urine, blood, pleural fluid, and on formalin-fixed paraffin-

embedded tissues. Using a clinical diagnosis as the gold standard for

tuberculosis diagnosis, the sensitivity of the PCR test is approximately

81% compared to AFB smear analysis (28%) and culture (63%). When a

clinical specimen is AFB-smear positive, the sensitivity of the

amplification method is approximately 95% with sensitivity 98%. (57-59)

When smear-negative specimens are examined, the sensitivity falls to

approximately 50%, but the specificity remains greater than 95%. For this

reason, these tests are recommended for smear-positive specimens only,

but their use in this regard is changing rapidly and varies from site to site.

Clearly, these methods have not replaced routine smear and culture.

Figure 15: PCR for Mycobacterium tuberculosis

Page 46: Tuberculosis. Pathogenesis and lab diagnosis

��

New Diagnostic Method for Tuberculosis

QuantiFERON®-TB gold (The Whole Blood IFN-gamma Test

Measuring Responses to ESAT-6 & CFP-10 Peptide Antigens)

1 – Intended used:

QuantiFERON®-TB Gold is an in vitro diagnostic test using peptide

cocktails simulating ESAT-6 and CFP-10 proteins to stimulate cells in

heparinised whole blood. Detection of interferon-�� (IFN-�) by Enzyme-

Linked Immunosorbent Assay (ELISA) is used to identify in vitro

responses to these peptide antigens that are associated with

Mycobacterium tuberculosis infection.

QuantiFERON®-TB Gold is an indirect test for M. tuberculosis infection

(including disease) and is intended for use in conjunction with risk

assessment, radiography and other medical and diagnostic evaluations.

2. Summary and explanation of the test:

The QuantiFERON®-TB Gold test is a test for Cell Mediated Immune

(CMI) responses to peptide antigens that simulate mycobacterial proteins.

These proteins, ESAT-6 and nCFP-10, are absent from all BCG strains

and from most non-tuberculosis mycobacteria with the exception of M.

kansasii, M. szulgai and M. marinum. Individuals infected with M.

tuberculosis complex organisms usually have lymphocytes in their blood

that recognise these and other mycobacterial antigens. This recognition

process involves the generation and secretion of the cytokine, IFN-�. The

detection and subsequent quantification of IFN-� forms the basis of this

test.

The antigens used in QuantiFERON®-TB Gold are a peptide cocktail

simulating the proteins ESAT-6 and CFP-10. Numerous studies have

demonstrated that these peptide antigens stimulate IFN-� responses in T-

cells from individuals infected with M. tuberculosis but generally not

from uninfected or BCG vaccinated persons without disease or risk for

LTBI. However, medical treatments or conditions that impair immune

functionality can potentially reduce IFN-� responses. Patients with certain

other mycobacterial infections might also be responsive to ESAT-6 and

CFP-10 as the genes encoding these proteins are present in M. kansasii,

M. szulgai and M. marinum. The QuantiFERON®-TB Gold test is both a

Page 47: Tuberculosis. Pathogenesis and lab diagnosis

��

test for LTBI and a helpful aid for diagnosing M. tuberculosis complex

infection in sick patients. A positive result supports the diagnosis of

tuberculosis disease; however, infections by other mycobacteria (e.g., M.

kansasii) could also lead to positive results. Other medical and diagnostic

evaluations are necessary to confirm or exclude tuberculosis disease.

3- Principles of the Assay

The QuantiFERON®-TB Gold assay detects CMI responses in vitro to

tuberculosis infection by measuring IFN-� in plasma harvested from

whole blood incubated with the TB and control antigens. The

QuantiFERON®-TB Gold test is performed in two stages.

First, four aliquots of herpanised whole blood are incubated with either

ESAT-6, CFP- 10, Mitogen or Nil control antigens.

Following 16 to 24 hours incubation, the plasma is removed and the

amount of IFN-� (IU/ml) measured by ELISA.

A test is considered positive for M. tuberculosis infection if they have an

IFN-� response to either ESAT-6 or CFP-10 that is significantly above

the Nil IFN-� IU/ml value.

The Mitogen-stimulated plasma sample serves as a positive control for

each individual tested. However, a positive response to either ESAT-6 or

CFP-10, without a response to Mitogen, is a valid result indicating

infection. A low response to Mitogen (<0.5 IU/ml) indicates an

indeterminate result when a blood sample also has a negative response to

the TB peptide antigens.

This pattern may occur with insufficient lymphocytes, reduced

lymphocyte activity due to improper specimen handling, or inability of

the patient’s lymphocytes to generate IFN-�.

The Nil samples adjust for background, heterophile antibody effects, and

non-specific IFN-� in blood samples. The IFN-� level of the nil samples

is subtracted from the IFN-� levels of the TB peptide antigens and

Mitogen control.

Page 48: Tuberculosis. Pathogenesis and lab diagnosis

��

*Time Required for Performing Assay:

The time required to perform the QuantiFERON®-TB Gold assay is

estimated below; the time of testing multiple samples when batched is

also indicated:

Blood collection: 2 to 5 minutes per sample

Blood culture set up: 10 minutes (add 1 to 1.5

* minutes per extra patient)

37°C Incubation of blood tubes: 16-24 hours

ELISA: Approx. 3 hours for one ELISA plate

• <1 hour labor

• Add 10-15 minutes for each *

extra plate

4- Reagents and storage:

4-1 Peptide and Control Antigens

1. Nil Control 1 x 6mL

2. ESAT-6 Peptides 1 x 6mL

3. CFP–10 Peptides 1 x 6mL

4. Mitogen Control 1 x 6mL

4-2 ELISA Components

1. Microplate strips 24 x 8 well

2. Human IFN-� Standard, lyophilised 1 x vial

3. Green Diluent 1 x 30mL

4. Conjugate 100X Concentrate, lyophilised 1 x 0.3mL

5. Wash Buffer 20X Concentrate 1 x 100mL

6. Enzyme Substrate Solution 1 x 30mL

7. Enzyme Stopping Solution 1 x l5mL

4-3 Storage Instructions

Peptide and Control Antigens • Store antigens at 2°C to 8°C.

• The shelf life of the QuantiFERON®-TB Gold antigens is 2 years from

the date of manufacture when stored at 2°C to 8°C.

Page 49: Tuberculosis. Pathogenesis and lab diagnosis

ELISA Kit Reagents • Store kit at 2°C to 8°C.

• Always protect Enzyme Substrate Solution from direct sunlight.

• The shelf life of the QuantiFERON®-TB Gold ELISA kit is 3 years

from the date of manufacture when stored at 2°C to 8°C.

5- Specimens collection and handling:

Completely fill a blood collection tube (minimum tube size 5mL)

containing heparin as the anticoagulant. Gently mix by inverting the tube

several times to dissolve the heparin, and transport to the laboratory at

ambient temperature (22°C ± 5°C).

Blood should be incubated with stimulation antigens as soon as possible

(as the IFN-� response decreases with time); and must be initiated within

12 hours of blood collection.

6. Directions for use:

6-1 Stage One – Incubation of Blood and Harvesting of Plasma

Refer to Section 4 for materials that are required when setting up

blood cultures. The TB peptides and control antigens do not need to be

brought to room temperature before use.

Procedure

1. Blood samples must be evenly mixed before aliquoting. Use a roller-

rocker or invert tubes 20 times immediately prior to dispensing.

2. Dispense 1.0mL aliquots (one per test antigen and control) of

heparinised whole blood from each subject into 4 wells of a 24 well tissue

culture plate (see Figure 17 for recommended layout). Blood is best

dispensed aseptically in a Biohazard cabinet using sterile pipettes to

minimise the risk of contamination.

3. Prior to use, mix each stimulation antigen well. Use undiluted. Holding

the dropper bottle vertically, carefully add 3 drops of each antigen to the

appropriate wells containing blood.

Page 50: Tuberculosis. Pathogenesis and lab diagnosis

FIGURE 16: Recommended layout for dispensing Blood and

Stimulation Antigens into 24 Well Culture Plates

4. Stimulation antigens must be mixed THOROUGHLY into the

aliquoted blood using a Microplate Shaker for 1 minute.

5. Incubate blood culture plates for 16-24 hours at 37°C in a humidified

atmosphere.

• Avoid stacking plates more than 2 high during incubation.

6. Carefully remove approximately 200-300�L of plasma from above the

sedimented red cells using a variable-volume pipette. Transfer the plasma

into separate 1 mL microtubes in a 96 well format or an empty 96 well

microtitre plate. Label sample racks appropriately.

• Use a new pipette tip for each plasma sample.

• Avoid harvesting blood cells with plasma. The assay will tolerate small

quantities of cells, but if the harvested plasma sample is grossly

contaminated with blood cells, centrifuge the sample to remove the cells.

7. Plasma can be stored at 2°C to 8°C for up to 28 days or at least 3

months at or below 20°C. Microtubes or microtitre plates should be

sealed appropriately prior to storage to avoid evaporation. Freezing at -

70°C is recommended to reduce the possibility of clot formation.

Page 51: Tuberculosis. Pathogenesis and lab diagnosis

��

6-2 Stage Two - Human IFN-� ELISA

Procedure 1. All plasma samples and reagents, except for Conjugate 100X

Concentrate, must be brought to room temperature (22°C ± 5°C) before

use. Allow at least 60 minutes for equilibration.

2. Remove strips that are not required from the frame, reseal in the foil

pouch, and return to the refrigerator for storage until required.

Allow at least one strip for the QuantiFERON®-TB Gold Standards and

sufficient strips for the number of subjects being tested.

After use, retain frame and lid for use with remaining strips.

3. Reconstitute the freeze dried Kit Standard with the volume of

deionised or distilled water indicated on the label of the Standard vial.

Mix gently to minimise frothing and ensure complete solubilisation.

Reconstitution of the Standard to the stated volume will produce a

solution with a concentration of 8.0 IU/mL.

Note: The reconstitution volume of the Kit Standard will differ

between batches. Use the reconstituted Kit Standard to produce a 1 in 4 dilution series of

IFN-� in Green Diluent (GD). S1 (Standard 1) contains 4 IU/mL, S2

(Standard 2) contains 1 IU/mL, S3 (Standard 3) contains 0.25 IU/mL, and

S4 (Standard 4) contains 0 IU/mL (GD alone). The standards should be

assayed at least in duplicate.

RECOMMENDED PROCEDUREFOR DUPLICATE

STANDARDS

a. Label 4 tubes “S1”, “S2”, “S3”, “S4”.

b. Add 150�L of GD to S1, S2, S3, S4.

c. Add 150�L of the Kit Standard to S1 and mix thoroughly.

d. Transfer 50�L from S1 to S2 and mix thoroughly.

e. Transfer 50�L from S2 to S3 and mix thoroughly.

f. GD alone serves as the zero standard (S4).

• Prepare fresh dilutions of the Kit Standard for each ELISA session.

Page 52: Tuberculosis. Pathogenesis and lab diagnosis

��

FIGURE 17: Preparation of Standard Curve

4. Reconstitute freeze dried Conjugate 100X Concentrate with 0.3mL of

deionised or distilled water. Mix gently to minimise frothing and ensure

complete solubilisation of the Conjugate.

Working Strength conjugate is prepared by diluting the required amount

of reconstituted Conjugate 100X Concentrate in Green Diluent as set out

in Table 2 - Conjugate Preparation.

Number Of Strips Volume Of

Conjugate 100X

Concentrate

Volume of Green

Diluents

2 10 ul 1.0 ml

3 15 ul 1.5 ml

4 20 ul 2.0 ml

5 25 ul 2.5 ml

6 30 ul 3.0 ml

7 35 ul 3.5 ml

8 40 ul 4.0 ml

9 45 ul 4.5 ml

10 50 ul 5.0 ml

11 55 ul 5.5 ml

12 60 ul 6.0 ml

• Mix thoroughly but gently to avoid frothing.

• Return any unused Conjugate 100X Concentrate to 2°C to 8°C

immediately after use.

• Use only Green Diluent.

Page 53: Tuberculosis. Pathogenesis and lab diagnosis

��

5. Prior to assay, plasmas should be mixed to ensure that IFN-� is evenly

distributed throughout the sample.

6. Add 50�L of freshly prepared Working Strength conjugate to the

required ELISA wells using a multichannel pipette.

7. Add 50�L of test plasma samples to appropriate wells using a

multichannel pipette (Refer to recommended plate layout below – Figure

19). Finally, add 50�L each of the Standards 1 to 4.

FIGURE 18: Recommended Sample Layout

• S1 (Standard 1), S2 (Standard 2), S3 (Standard 3), S4 (Standard 4).

����������������������� ������!��"#��$����mple 1 ESAT-%����!��"#����(Sample 1 CFP-�&����!��"#��'�����������'���(������� ������!��")

8. Mix the conjugate and plasma samples/standards thoroughly using a

microplate shaker for 1 minute.

9. Cover each plate with a lid and incubate at room temperature (22°C ±

5°C) for 120 ± 5 minutes.

• Plates should not be exposed to direct sunlight during incubation.

10. During the incubation, dilute one part Wash Buffer 20X Concentrate

with 19 parts deionised or distilled water and mix thoroughly. Sufficient

Wash Buffer 20X Concentrate has been provided to prepare 2L of

Working Strength wash buffer.

Page 54: Tuberculosis. Pathogenesis and lab diagnosis

��

Wash wells with 400�L of Working Strength wash buffer for at least 6

cycles. An automated plate washer is recommended.

• Thorough washing is very important to the performance of the assay.

Ensure each well is completely filled with wash buffer to the top of the

well for each wash cycle. A soak period of at least 5 seconds between

each cycle is recommended.

• Standard laboratory disinfectant should be added to the effluent

reservoir, and established procedures followed for the decontamination of

potentially infectious material.

11. Tap plates face down on absorbent towel to remove residual wash

buffer. Add 100�L of Enzyme Substrate Solution to each well and mix

thoroughly using a microplate shaker.

12. Cover each plate with a lid and incubate at room temperature (22°C ±

5°C) for 30 minutes.

• Plates should not be exposed to direct sunlight during incubation.

13. Following the 30 minute incubation, add 50�L of Enzyme Stopping

Solution to each well and mix.

• Enzyme Stopping Solution should be added to wells in the same order

and at approximately the same speed as the substrate in step 11.

14. Measure the Optical Density (OD) of each well within 5 minutes of

stopping the reaction using a microplate reader fitted with a 450nm filter

and with a 620nm to 650nm reference filter. OD values are used to

calculate results.

Page 55: Tuberculosis. Pathogenesis and lab diagnosis

��

7- Interpretation of results:

QuantiFERON®-TB Gold results are interpreted using the following

criteria:

1 Responses to the Mitogen positive control (and occasionally ESAT-6

and/or CFP-10) can be commonly outside the range of the microplate

reader. This has no impact on test results.

2 Where M. tuberculosis infection is not suspected, initially positive

results can be confirmed by retesting the original plasma samples in

duplicate in the QuantiFERON®-TB Gold ELISA. If repeat testing of

one or both replicates is positive, the individual should be considered test

positive.

3 Refer to Section 9 for possible causes.

4 In clinical studies, less than 0.25% of subjects had IFN-� levels of > 8.0

IU/mL for the Nil Control.

The magnitude of the measured IFN-� level cannot be correlated to stage

or degree of infection, level of immune responsiveness, or likelihood for

progression to active disease.

Page 56: Tuberculosis. Pathogenesis and lab diagnosis

��

8. Warnings and precautions:

8-1 Warnings

• A negative QuantiFERON®-TB Gold result does not preclude the

possibility of M. tuberculosis infection or tuberculosis disease: false-

negative results can be due to stage of infection (e.g., specimen obtained

prior to the development of cellular immune response), co-morbid

conditions which affect immune functions, incorrect handling of the

blood collection tubes following venipuncture, incorrect performance of

the assay, or other immunological variables.

• A positive QuantiFERON®-TB Gold result should not be the sole or

definitive basis for determining infection with M. tuberculosis. Incorrect

performance of the assay may cause false positive responses.

• A positive QuantiFERON®-TB Gold result should be followed by

further medical evaluation and diagnostic evaluation for active

tuberculosis disease

(e.g., AFB smear and culture, chest x-ray).

• While ESAT-6 and CFP-10 are absent from all BCG strains and from

most known non-tuberculous mycobacteria, it is possible that a positive

QuantiFERON®-TB Gold result may be due to infection by M. kansasii,

M. szulgai or M. marinum. If such infections are suspected, alternative

tests should be investigated.

8-2 Precautions

• For in vitro diagnostic use.

• Harmful: Enzyme Substrate Solution contains 3,3_,5,5_

Tetramethylbenzidine that is harmful by ingestion, inhalation and skin

contact. Skin and eye irritant. Mutagen.

Use eye protection, wear gloves and handle as a potential carcinogen.

• Harmful: Enzyme Stopping Solution contains H2SO4 that is harmful

by ingestion, eye contact, skin contact, and inhalation. Use eye

protection, wear gloves and normal laboratory protective clothing. If the

stopping solution contacts the skin or eyes, flush with copious quantities

of water and seek medical attention.

Page 57: Tuberculosis. Pathogenesis and lab diagnosis

��

• Harmful: IFN-� Standard and Conjugate 100X Concentrate may be

discomforting if ingested and may cause skin irritation. Wear gloves and

normal laboratory protective clothing.

• Handle human blood as if potentially infectious. Observe relevant

blood handling guidelines.

• Thimerosal is used as a preservative in some reagents. It may be toxic

upon ingestion, inhalation or skin contact.

• Green Diluent contains normal mouse serum and casein, which may

trigger allergic responses; avoid contact with skin.

• Deviations from the directions for use in the Package Insert may yield

erroneous results. Please read the instructions carefully before use.

• Do not use kit if any reagent bottle shows signs of damage or leakage

prior to use.

• Do not mix or use ELISA reagents from other QuantiFERON®-TB

Gold kit batches.

• Discard unused reagents and biological samples in accordance with

Local, State, and Federal regulations.

• Do not use the TB peptides and control antigens or ELISA kit after the

expiry date. (60)

Page 58: Tuberculosis. Pathogenesis and lab diagnosis

��

��������������

������ ����

Page 59: Tuberculosis. Pathogenesis and lab diagnosis

BCG Vaccine of Tuberculosis

1: BCG Vaccine

BCG vaccine is a live attenuated vaccine derived originally from

mycobacterium Bovis cultured on different media for hundreds of times

for many years.

As the manufacturer companies are using different methods for

preparation of BCG from the mother strain (Pasteur strain), the

immunogenicity differs from one strain to another. There are many

strains for BCG vaccine like Danish, Japanese and Glaxo.

BCG vaccine is considered as the gate for other vaccines in kingdom,

as it is administered at birth, it has to be administered carefully with all

possible precautions.

2: Storing and validity

* The vaccine should be stored at 2-8 C in the refrigerator

* The vaccine should not be used after the expiry date shown on the

bottle.

* It should be kept out of direct sun light.

* It should be used within four hours after dilution.

* The manufacturer's instructions accompanied with the vaccine should

be strictly followed.

3: Indications

BCG vaccination protects from the sever forms of tuberculosis and has

to be given to the following groups:

• Infants at birth.

• Tuberculin negative contacts of pulmonary smear positive patients.

• Children who are not previously vaccinated.

• Children previously vaccinated but after three month does not

develop BCG scar and remain tuberculin negative.

Page 60: Tuberculosis. Pathogenesis and lab diagnosis

4: Complications

• Skin ulcer of the site of administration.

• Localized lymphoadenopathy.

• Localized abscess.

These complications are usually mild and need no treatment except for

cleaning and disinfection. In case of sever complications the following

should be done:

• Clean and disinfect the site of vaccination.

• Aspirate the abscess or open it surgically.

• Apply antibiotic locally systemically.

5- Contraindications

• Fever.

• Pregnancy.

• Children with HIV infection.

• Hereditary immunodeficiency.

• Extensive skin inflammatory conditions and burn.

• Children under immunosuppressive therapy.

6- BCG vaccination and other vaccines

BCG vaccine can be administered in the same setting with other

vaccines (DPT, Polio, and Measles). In case these vaccines are not

administered at the same setting, a period of three weeks has to elapse

before the following vaccination setting.

7- Administration and dosage

• 0.05 ml for neonates and children below one year of age (vaccine

has to diluted in two ml of diluent's and 0.1 ml of the diluted

vaccine is used).

• 0.1 ml for children more than one year of age (the vaccine has to be

diluted in one ml diluent's and 0.1 ml of the diluted vaccine is

used).

• Sterilized syringe with 26 mm has to be used.

Page 61: Tuberculosis. Pathogenesis and lab diagnosis

��

8- Site

a- Administration should be intradermal at the external part of the left

deltoid muscle.

b- Alcohol should not be used for the purpose of disinfection of the

vaccination site.

c- If the vaccine was correctly administered, erythema will occur at the

site of administration followed by pustule formation containing yellow

fluid which then dries leaving a permanent scar. (42)

Figure 19: BCG vaccine

Page 62: Tuberculosis. Pathogenesis and lab diagnosis

��

������������

������ ����

Page 63: Tuberculosis. Pathogenesis and lab diagnosis

��

TREATMENT of Tuberculosis

1- General role of tuberculosis treatment

Tuberculosis treatment must be started before confirmation of

diagnosis. It has started after receiving laboratory reports at least two

smear positive sputum samples. In case of the presence of only one

positive sputum sample, the decision of treatment should be taken by

the physician. In absence of positive laboratory smear results, the

decision of treatment should be taken by the physician guided by

clinical and X-ray findings and at least two sputum samples negative

by microscopy for acid fast bacilli. In treatment of tuberculosis the

following should be put in mind.

• Anti-tuberculosis drugs should be used according to the

recommended categories.

• For the recommended period.

• And under direct supervision.

2- Directly Observed Treatment, Short course (DOTS)

DOTS is considered to be the optimal way for treating tuberculosis

patients because of the following:

• Short duration of treatment helps patients to adhere to treatment.

• Rapid conversion of sputum from positive to negative decreases

the chance of infection transmission.

• The high rate compared with low cost.

• Decreased complications of tuberculosis.

• Decreasing the chance of emergence of drug resistant tuberculosis.

• Decreasing mortality rate.

3- Phases of treatment

The period of tuberculosis treatment is derived into two phases: an

initial intensive phase for a period not less than two months where 3-4

drugs are used and a continuation phase for a period not less than four

months where at least two drugs are used. The use of this combination of

drugs in the intensive phase, including refampicin, help to eliminate

tuberculosis bacilli from the body and decrease the chance of emergence

of resistant strain. It is recommended to extend the intensive phase by one

month if sputum remains positive by the end of the second month of

treatment in new cases and end of the third month in re-treatment cases.

Page 64: Tuberculosis. Pathogenesis and lab diagnosis

��

4- Hospitalization

It is important to hospitalize pulmonary smear positive tuberculosis

patients during the intensive phase of treatment (two months or more).

Also, critical cases, complicated cases and some other should be

hospitalized if the physician recommends that.

5- Duration of treatment

The duration of treatment should be not less than 6 months and there is

no need for expansion of this period if the patient adheres to treatment,

except in some exceptional cases.

6- General procedures that should be followed during treatment

The patient should be followed-up during the period of treatment to

ensure his adherence to treatment and to perform follow-up should be for

pulmonary smear positive patients where sputum must be examined at the

end of the second month of treatment (third month in case of relapse or

failure), fifth month and at the end of treatment.

If smear remains positive by the end of the second month (third month

in case of relapse or failure), the intensive phase should be extended for

another month (third month in new cases and forth month in relapse or

failure cases) till the sputum converted to negative then the continuation

phase has to be started.

If smear remains positive by the end of the third month (fourth month

in relapse and failure cases), the treatment must be stopped for three days

and a sputum sample should be examined by culture and sensitivity then

the continuation has to be continued to the fifth month.

If sputum remains positive by the fifth month the patient has be

reregistered as a failure case.

If the patient was initially pulmonary smear negative and by the end of

the second month of treatment converted to positive, he should also

reregistered as a failure case.

Page 65: Tuberculosis. Pathogenesis and lab diagnosis

��

7- Categories of treatment

The following are the main drugs used in treatment of tuberculosis and

their codes:

• Isoniazid (H)

• Rifampicin (R)

• Pyrazinamide (Z)

• Ethambutol (E)

• Streptomycin (S)

- Anti-tuberculosis drugs should be reviewed regularly (every 6-8

months) for quality and expiry date.

- The period of validity for these drugs from the date of production

is as follow:

• Isoniazid (5 years).

• Refampicin (3 years).

• Pyrazinamide (3 years).

• Ethambutol (5 years).

• Streptomycin ( 3 years).

- The use of anti-tuberculosis drugs, especially refampicin and

streptomycin, should be very limited in treatment of diseases other

than tuberculosis.

- Treatment codes:

2HRZE/4HR means that four drugs are taken daily for two months in

the intensive phase and two drugs are taken daily for four months in

the continuation phase.

7-1 CAT1 [2HRZS(E)/2HR]: This category is administered to new smear positive cases, sever

pulmonary smear negative patients (as extensive parenchymal

involvement) and sever extra-pulmonary forms (meningitis,

pricarditis, miliary and peritoneal tuberculosis).

- Intensive phase [2HRZS(E)]: Four anti-tuberculosis drugs

[Isoniazid, refampicin, pyrazinamide, streptomycin (Ethambutol)]

are administered daily for two months. If sputm converted to

negative the continuation phase should be started, otherwise the

intensive phase must be extended for another month.

Page 66: Tuberculosis. Pathogenesis and lab diagnosis

��

- Continuation phase 94HR0: Started after conversion of sputum

from positive to negative or if the sputum remains positive after

expansion of intensive phase to a third month (in this case

treatment must be stopped for three days and sputum samples for

culture and sensitivity must be collected) and two drugs [Isoniazide

and Refampicin (Ethmbutol)] are used daily for four months.

7-2 CAT2 [2HRZSE/1 HRZE/5HRE]: This category of treatment as administered to cases classified as

relapse and failure of treatment. Drug resistant tuberculosis should be

suspected in these cases. In patients who had previously treated from

tuberculosis for more than one month, treatment must be stopped for

three days and a sputum sample for culture and sensitivity must be

collected before starting the new category of treatment then drugs must

be used in accordance with the results of culture and sensitivity. A

competent follow-up for those patients is recommended to ensure their

adherence to treatment because the chance of having drug resistant

tuberculosis is being high.

- Intensive phase [2 HRZSE/1 HRZE]: Five drugs [Isoniazide,

Refampicin, Pyrazinamide, Streptomycin and Ethmbutol] are used

daily for two months and then four drugs daily [all drugs

mentioned above except for Streptomycin] for one month. If

sputum remains positive by the end of the third month, the

intensive phase must be extended for a four month. If sputum

remains positive by the end of the fourth month, treatment must be

stopped for three days and sputum samples for culture and

sensitivity must be collected.

- Continuation phase [ 5 HRE]: Three drugs (Isoniazide, Refampicin

and Ethmbutol0 are used for five months and if the intensive phase

was extended for a fourth month the continuation phase must be

extended for a sixth month).

7-3 CAT3 [2 HRZ/ 4 HR or 6HE]: This category is administered for patients with new smear negative

pulmonary tuberculosis (not sever), extra-pulmonary (not critical) and for

children complaining of tuberculosis.

- Initial intensive phase [2 HRZ]: three drugs (isoniazide,

Refampicin and Pyrazinamide) are used daily for two months.

- Continuation phase (4HR or 6HE): Isoniazide and Refampicin are

used daily for four months or Isoniazide and Ethmbutol are used

daily for six months.

Page 67: Tuberculosis. Pathogenesis and lab diagnosis

��

7-4 CAT4: This is special category of treatment used for chronic and resistant

tuberculosis case. Treatment must be based on culture and sensitivity and

usually the second line drugs are used for treatment of those patients. (42)

Page 68: Tuberculosis. Pathogenesis and lab diagnosis

��

������ ������

������ ����

Page 69: Tuberculosis. Pathogenesis and lab diagnosis

Extrapulmonary Tuberculosis

The classic definition of extrapulmonary TB is the tuberculosis

involvement of an organ outside of the lung. The course of

extrapulmonary TB may be acute and overwhelming, or chronic and

slowly progressive over many years, and any organ may be involved.

1- Hepatic TB

The liver may be involved in all forms of tuberculosis, including

pulmonary, extrapulmonary, and miliary or disseminated disease.

Noncaseating hepatic granulomas have been described in 25% of patients

with pulmonary tuberculosis without evidence of clinical hepatitis. (61)

Percutaneous liver biopsies may show granulomata in 50 to 100% of

patients with miliary tuberculosis. (62, 63, 64) Liver involvement usually

occurs with dissemination, but can be an isolated process in 5% of

patients. (62) Hepatic tuberculosis may be seen with granulomatous

disease, isolated or multiple abscesses, fibrosis. cirrhosis, or chronic

hepatitis. (62)

1-1Clinical:

Hepatic tuberculosis may be asymptomatic or manifest with fever,

right upper quadrant pain, or jaundice and may mimic a variety of

conditions from infections to neopalsms. (62) Often there are no localizing

symptoms. In one study, 10% of patients with clinically unexplained

hepatomegaly had tuberculosis. (65) The alkaline phosphates is elevated

with space-occupying granulomas in 30% of patients, transaminases often

are normal, and hyperbilirubinemia is minimal or absent. (62)

Hepatomegaly is present in 50% of patients and splenomegaly in 30% of

patients with hepatic granulomas. (66)

Page 70: Tuberculosis. Pathogenesis and lab diagnosis

Figure 20: Hepatic tuberculosis

1-2Diagnosis:

The diagnosis can be established by sonogram-guided percutaneous

biopsy in 70% of patients or laparoscopy in 90% of patients, although

stains are negative in 50 to 90% of cases. (66) The sonogram may show

periportal, lymph nodes that potentially may obstruct the biliary system.

(67) Computed tomography of the abdomen may show hepatomegaly or a

mass lesion. (68)

Surgery is indicated for diagnosis and possibly for abscess drainage.

(69) Mortality due to hepatic tuberculosis is related to respiratory

insufficiency, peritonitis, portal vein thrombosis, and portal hypertension

with variceal hemorrhage. (64)

Page 71: Tuberculosis. Pathogenesis and lab diagnosis

��

2- Meninges:

Neurotuberculosis is a life threatening complication which can affect

all regions of the CNS, although there is a predilection for the basilar

meninges. Tuberculosis of the nervous system can affect the meninges,

brain, spinal cord, cranial and peripheral nerves, ears, and eyes. (69)

Tuberculous meningitis may occur at any age, but historically is a

disease of children in the first 5 years of life. It is uncommon in children

less than 6 months of age and rare under the age of 3 months. (70)

Figure 21: Meningeal tuberculosis

2-1Clinical:

Meningitis may develop either from a local activated dormant focus or

from a distant site, e.g., lung or paravertebral abscess, through

hematogenous spread, or as the result of miliary tuberculosis. Regardless

of the site of region, there must be rupture of a caseous focus into the

arachnoid space. This focus usually lies adjacent to the meninges. (71)

Pathologically, only a small number of living bacilli may be seen in

the parenchyma, and serous meningitis may result without evidence

organisms. Hyperemia, capillary damage, scars, and edema are observed.

A thick gelatinous exudates may collect at the base of the brain,

Page 72: Tuberculosis. Pathogenesis and lab diagnosis

��

interfering with cranial nerve function, and hydrocephalus may occur.

Basal meningeal inflammation may spread focally to adjacent tissues.

Granulomas are seen within the choroids plexus in 75% of cases and the

ependyma in 90% of cases. Small discrete gray white tubercles may

visible over the entire surface of the brain. (72)

The onset of tuberculous meningitis is insidious with a 2-week

prodromal period before meningeal symptoms occur. (73) The clinical

features include fever, anorexia, malaise, nausea, vomiting, headache,

apathy, and mental alterations. Physical finding include nuchal rigidity,

basilar cranial nerve involvement, focal neurologic deficits, pupillary

changes, funduscopic changes, papilledema, and peripheral adenopathy.

(74) A waxing and waning course with sudden acceleration may occur,

especially in children. (72)Non neurologic tuberculosis is associated with

37% of patients. (75)

Most cases of tuberculosis meningitis progress through three stages.

The first stage occurs with low grade fever, personality changes, and

irritability, and lasts 1 to 2 weeks. Confusion may be an early sign in the

elderly. The cerebrospinal fluid (CSF) shows few neutrophils and a

borderline normal glucose and protein. During the second stage there is

an increase in the intracranial pressure with nausea, vomiting nuchal

rigidity, photophobia, seizures, and cranial nerve palsies are seen. The

CSF shows lymphocytes, an increased protein and decreased glucose

content. The last stage is associated with high fever, confusion, stupor,

and coma. Decortication and herniation eventually may ensue. (76)

2-2Diagnosis:

The tuberculin skin test can be negative in as much as 50% of cases,

(77) but usually becomes positive during chemotherapy. (78) Hyponatremia

is common and usually is secondary to inappropriate secretion of

antidiuretic hormone. (73)

Examination of the CSF is the most valuable procedure in the

diagnosis of meningeal tuberculosis. The opening pressure during lumbar

puncture usually is elevated. The CSF protein is elevated and ranges from

100 to 500 mg/dl, and my rise considerably higher if there is a spinal

block with xanthochromia. (73, 20) an increasing protein concentration is

common during therapy and dose not necessarily portends treatment

failure. (75) The CSF glucose concentration declines in untreated cases.

Page 73: Tuberculosis. Pathogenesis and lab diagnosis

��

Intravenously administrated glucose should be avoided 2 hours before the

lumber puncture. (70)

The cellular changes in the CSF reflect a tuberculin reaction provoked

by the presence of tuberculoprotiens. There may be a moderate degree of

pleocytosis, usually with less than 500 cells/mm3 and rarely greater than

1200cells/mm3. More than 80 to 95% are lymphocytes, but a

predominance of polymorphonuclear cells may be seen early. (73)

The AFB smear of the CSF has been reported to be positive in 10 to

40% of specimens, (80) but the yield may increase to 85 to 87% with

repeated or centrifuged specimens. (81-83) Serial lumber punctures, even

after the onset of therapy, may contain the organisms. (84) The AFB smear

may be reported to be positive in 0 to 27% of patients with HIV infection.

(85) Tubercle bacilli may be isolated by culture in 50 to 80% of cases. (73)

Achieving the diagnosis at an early stage is critical but often onerous

because cerebrospinal fluid may be normal in as much as 20% of HIV-

positive patients. (75) If the CSF remains normal within 2 weeks of

presentation, the diagnosis of tuberculosis meningitis is unlikely. It may

be reasonable to withdraw treatment and follow the patient over the next

few months with repeat lumber punctures. (86)

More elaborate tests have been utilized in an attempt to establish the

diagnosis of tuberculous meningitis. Adenosine deaminase (ADA) is

elevated in the CSF and 60% of patients have levels greater than 10

IU/ml. Unfortunately ADA also may be increased in bacterial meningitis

and, therefore, is nondiagnostic. (87) In one study in HIV-infected patients,

the sensitivity of ADA was 63%, better than the AFB smear. (75) Enzyme-

linked immunoabsorbent assay (ELISA) to detect soluble mycobacterial

antigen (e.g., antigen 5) may be useful, with a sensitivity of 80% and a

specificity nearing 100%. (88-90) An immunoblot technique using

mycobacterial antigen 60 (A60) has been developed. The early

appearance of antimycobacterial immunoglobulins to this complex may

be seen in the CSF of patients with tuberculous meningitis. (91)

Detection of tuberculostearic acid, a fatty acid resent only in

mycobacteria, provides a diagnostic test with a high degree of accuracy.

This may present the best approach to the rapid diagnosis of tuberculous

meningitis. (92)

The bromide partition test and lactate levels are less useful, but the

polymerase chain reaction (PCR) technique can yield a sensitivity above

*+,#� !��-�.�-��/� �!� ��0� )� 1�2� �� .� ��3� ��� !�� �3� ��4� ��- ��!�!�specificity by decreasing possible laboratory contamination. (93)

Page 74: Tuberculosis. Pathogenesis and lab diagnosis

��

Multicenter trials would be most helpful in establishing the clinical

usefulness of these various tests. (94) A meningeal biopsy or brain biopsy

may be necessary, on occasion, but carries significant risk, including

postoperative epidural hematoma and hydrocephalus. (95)

Page 75: Tuberculosis. Pathogenesis and lab diagnosis

��

Conclusion

Pulmonary Tuberculosis is a disease caused by germ called

Mycobacterium tuberculosis, its affect primary the lung, and cause

cavitations in the lung, and can affect another sites of body.

Mycobacterium tuberculosis can stills in the host of body for long

time, until the conditions of growth occur.

The diagnosis is depends on the laboratory tests, sputum examination

and culture (other specimen for another sites of infection), PCR.

There is a new test to detect tuberculosis depends on IFN-gamma

measures, and its take short time when compared with old tests.

The tuberculosis patient must be isolates in hospital for the first two

months from beginning of treatment, and the treatment takes time 6-8

months.

The vaccine give immunity up to 80%, the most active control for this

disease is the healthy education.

Page 76: Tuberculosis. Pathogenesis and lab diagnosis

��

REFERENCES 1-Geo. F. Brooks , Janet S. Butel , & Stephen A. Morse said in Jewetz , Melnick, &

Adelberg's, 2001, Medical Microbiology twenty-third edition 319, 321.

2- Medical Microbiology third edition, 1991, 453, 455.

3- Noll, H., 1956, The chemistry of card factor, a toxic glycolipid of M. tuberculosis,

Adv. Tuberc. Res., 7, 149.

4-Bloch, H., 1950, Studies on the virulence of tubercle bacilli, J. Exp. Med., 91, 197.

5-Artman, M., Bekierkunst A., and Goldenberg, I., 1964, Tissue metabolism in

Infection: Biochemical changes in mice treated with cord factor, Arch. Biochem.

Biophys., 105, 80.

6-Goren, M.B., 1970, Sulfolipid I of Mycobacterium tuberculosis strain H37Rv. II.

Structural studies, Biochem. Biophys. Acta, 210, 127.

7-Gordon, A.H., D'Arcy Hart, P., and Young, M.R., 1980, Ammonia inhibits

phagosome-lysosome fusion in macrophage. Nature (London), 286, 79.

8- Goren, M.B., 1970, Sulfolipid I of Mycobacterium tuberculosis strain H37Rv. II.

Structural studies, Biochem. Biophys. Acta, 210, 127.

9-Brennan, P.J. and Draper, P., 1970, Ultrastructure of Mycobacterium tuberculosis,

In Tuberculosis: Pathogenesis, Protection, and control, (B. Bloom, Ed.), ASM Press,

Materials Park, OH.

10-Goren, M. B., D'Arcy Hart, P., Young, W. R., and Armstrong, J. A., 1976,

Prevention of phagosome-lysosome fusion in cultured macrophage by sulfatides of

Mycobacterium tuberculosis, Proc. Nat Acad. Sci. USA, 73, 2510.

11- Smith, D.W., Randall, H.M., Gaastambide-Odier, M.D., and Koevote, A.L., 1960,

Mycosides: a new class of type-specific glycolipid of mycobacteria, Ann. N.Y. Acad.

Sci., 69, 145.

12- Brennan, P.J., Hunter, S.W., Mcneil, M., Chatterjee, D., and Daffe, M., 1900,

Reappraisal of the chemistry of mycobacterial cell walls, with a view to

understanding the roles of individual entities in disease processes, In Microbial

Determinants of Virulence and Host Response, Ayoub, E.M., cassell, G.H., Branch,

W.C., Jr., and Henry, J.T., Eds., American Society for Microbiology, Washington,

D.C.

13- Rastogi, N., 1991, Recent observation concerning structure and function

relationship in the mycobacterial cell envelope: elaboration of a model in terms of

microbial pathogenicity, virulence and drugresistance, res. Microbial., 142, 464.

14- Gomez, J.E., Chen, J-M., and Bishai, W.R., 1997, Sigma factors of

mycobacterium tuberculosis, Tuberc. Lung Dis., 78, 175.

Page 77: Tuberculosis. Pathogenesis and lab diagnosis

��

15- Cole E, Cook C, 1998, Characterization of infectious aerosol in health care

facilities: an aid to effective engineering controls and preventive strategies.

16- World Health Organization (WHO) march 2000.

17- Griffith D, Kerr C, 1996, Tuberculosis: disease of the past, disease of the present.

2003

18- Centers for Disease Control and Prevention (CDC), Division of Tuberculosis

Elimination. Core curriculum on Tuberculosis: What the clinical Should Know. 4th

edition 2000.

19- David Greenwood , Richard C.B. Slack and Jhon F. Peutherer., 2002 Medical

Microbiology sixteenth edition.

20- Kaufmann SH., 2005, Recent findings in immunology give tuberculosis vaccines

����0�5��!�)�6 ��3!�7��8���)#�9%��9":%%&-7.

21- North RJ, Jung YJ, 2004, Immunity to Tuberculosis. Annue Rev Imminol.;(

22):599-623.

22- Winau F, Weber S, Sad S, de Diego J, Hoops SL, Breiden B. 2006, Apoptotic

ve!�-��� - �!!� ���� �;<� 6� -���!� ��3� � ���-�� �(���!�� �85� -8��!�!)� 7��8���/)#�24(1):105-17.

23- Guermonprez P, Saveanu L, Kleijmeer M, Davoust J, Van Endert P, Amigorena

S., 2003, ER-phagosome fusion defined an MHC class � cross-presentation

compartment in de�3 ���-�-���!)����8 �)#�=9+�%>+%":?>*-402.

24- Tanaka Y, Morita CT, Tanaka Y, Nieves E, Brenner MB, Bloom BR., 1995,

Natural and synthetic non-peptide antigens recognized by human gamma delta T cells.

���8 ��)#�?*+�%+9*":�++-8.

25- Grant EP, Degano M, Rosat JP, Stenger S, Modlin RL, Wilson LA., 1999,

'���-8�� � �-�(��������.� ����3�����(��!�5/�6�-���� �-���� !)� @�$A��'�3)#�<>��":�>+-

205.

26- Kaufmann SH, Schaible UE., 2005, Antigen presentation and recognition in

bacterial infections. Curr Opin Immunol.; 17(1):79-87.

27- Teixeria HC, Munk ME, Kaufmann SH., 1995, Frequencies of IFN gamma- and

IL-4-production cells during Mycobacterium bovis (BCG) infection in two genetically

susceptible mouse strains: role of alpha/beta T cells and NK1.1 cells, Immunol left.;

46(1-2):338-44.

28- Medzhitov R, Janeway C Jr., 2000, 7������7��8���/)���$�(��@�'�3)#�?=?�+":??<-

44.

29- Ottenhoff TH, Verreck FA, Hoeve MA, van de Vosse E., 2005, Control of human

B�!�����8���/�����/-�5�-�� ��)�685� -8��!�!��$3��5")#�<+��-2):53-64.

Page 78: Tuberculosis. Pathogenesis and lab diagnosis

��

30- Ottenhoff TH, Kumararatne D, Casanova JL., 1998, Novel human

immunodeficiencies reveal the essential role of type-1 cytokines in immunity to

��� �-���8�� �5�-�� ��)�7��8����6�3�/)#��>���":=>�-4.

31- Salgame P., 2005, Host innate and th1 responses and the bacterial factors that

-��� ���'/-�5�-�� �8���85� -8��!�!���.�-����)��8 �C����7��8���)#�*�=":?*=-80.

32- Jouangruy E, Altare F, Lahmamedi S, Revy P, Emile JF, Newport M., 1996,

Interferon-gamma-receptor deficiency in an infant with fatal bacille Calmette-Guerin

��.�-����)���$�(��@�'�3)#�??+�9%"#�>%+-61.

33- Lin Y, Zhang M, Hofman Fm, Gong J, Barnes PF., 1996, Absence of a prominent

6B9�-/��D���� �!���!�����B8�����85� -8��!�!)#�%=�=":�?�+-6.

34- Swaminathan S, Gong J, Zhang M, Samten B, Hanna LE, Narayanan PR., 1999,

Cytokine production in children with tuberculosis infection and disease. Clin Infect

;�!)#�9<�%":�9>&-3.

35- Ting LM, Kim AC, Cattamanchi A, Ernst JD., 1999, Mycobacterium tuberculosis

inhibits IFN-gamma transcriptional responses without inhibiting activation of STAT1.

@�7��8���)#��%?�*"?<><-906.

36- Boehm U, Klamp T, Groot M, Howard JC., 1997, Cellular responses to

interferon-(����)�E��8�7��8���)#��+:*=>-95.

37- Seah GT, Scott GM, Rook GA., 2000, Type 2 cytokine gene activation and its

relationship to extent of disease in patients with tuberculosis. J Infect Dis.;

181(1):386-9.

38- Rook GA, Hernandez-Pando R, Dheda K, Teng Seah G., 2004, IL-4 in

�85� -8��!�!:������-�����!�.� �F�--����3�!�(�)�6 ��3!�7��8���)#�9+�>":=<?-8.

39- Rhoades ER, Cooper AM, Orme IM., 1995, Chemokine response in mice infected

0��B�'/-�5�-�� �8���85� -8��!�!)�7�.�-��7��8��)#�%?��&":?<*�-7.

40- Brice GT, Graber NL, Hoffman SL, Doolan DL., 2001, Expression of the

chemokine MIG is a sensitive and predictive marker for antigen-specific, genetically

restricted IFN-gamma production and IFN-gamma-screeting cells. J Immunol

'��B�3!)#�9+*��-2):55-69.

41- Abramo C, Meijgaarden KE, Garcia D, Franken KL, Klein MR, Kolk AJ., 2006,

Monokine induced by interferon gamma and IFN-gamma response to a fusion protein

of Mycobacterium tuberculosis ESAT-6 and CFP-10 in Brazilian tuberculosis

�������!)�'�- �5�!�7�.�-�)#�<��":=+-51.

42- Nbil AL-kahtani Consultant of Preventive Medicine and National Coordinator of

NTP, Mohammad AL-Jeffri Director General of Parasitic & Infectious Disease.,

2003, Manual Of The National Tuberculosis Control Program, 29, 78.

43- http://www.healthatoz.com/

Page 79: Tuberculosis. Pathogenesis and lab diagnosis

44- Mullis. K. B. and Faloona. F., 1997, Specific synthesis of DNA in vitro via a

polymerase catalyzed chain reaction. Meth. Enzymol., 35, 907.

45- Hance, A. J., Grandchamp, B., Lavy-Frebault, V., Lecossier, D., Rauzier, J.,

Bocart, D., and Gicqual, B., 1990, Detection of mycobacteria by amplification of

mycobacterial DNA, Mol. Microbial,. 3, 1877.

46- Manjunath. N., 1991, Evaluation of a polymerase chain reaction for the diagnosis

of tuberculosis, Tubercle, 72, 21.

47- Pao, C. C., Yen, T. S. B., You, J. B., Maa, J. S., Fiss, E. H., and cahang, C. H.,

1990, Detection and identification of Mycobacterium tuberculosis by DNA

amplification, J. Clin. Microbial,. 28, 1877.

48- Shankar, P., Manjunath, N., Lakshmi, R., Aditi, B., Seth, P., and Shriniwas, K.,

1990, Identification of Mycobacterium tuberculosis by polymerase chain

reaction, Lancet, 355, 423.

49- Sjobring, U., Mecklenburg, M., Anderson, A. B., and Miorner, M., 1990,

Polymerase chain reaction for detection of Mycobacterium tuberculosis, J. Clin.

Microbial., 28, 2200.

50- Plikaytis, B. B., Eisenach, K. D., Crawford, J. T., and Shinnick, T. M., 1991,

Differentiation of Mycobacterium tuberculosis and Mycobacterium bovis by a

polymerase chain reaction assay, Mol. Cell Probes, 5, 215.

51- Boddinghaus, B., Rogall, T., Flohr, T., Blocker, H., and Bottger, E. C., 1990,

Detection and identification of mycobacteria by amplification of rRNA, J. Clin.

Microbial., 28, 1751.

52- Eisenach, K.D., Cave, M. D., Bates, J. H., and Crawford, J, T., 1990, Polymerase

chain reaction amplification of a repetitive DNA sequence specific for

Mycobacterium tuberculosis, J. Infect. Dis., 161, 977.

53- Thierry, D., Cave, M. D., Eisenach, K.D., Bates, J. H., Gicqual, B., and Guesdon,

T. L., 1990, IS6110 and IS-like element of Mycobacterium tuberculosis

complex, Nucl. Acids Res., 18, 188.

54- Eisenach, K. D., Sifford, M. D., Bates, J. H., and Crawford, J. T., 1991, Detection

of Mycobacterium tuberculosis in sputum using a polymerase chain reaction,

Am. Rev. Resp. Dis., 144, 1160.

55- Centers for Disease Control and Prevention, 1996, Nucleic acid amplification

tests for tuberculosis, MMWR, 45, 950.

56- Cohen, R., Muzaffar, S., Schwartz, D., Bashir, S., Luke, S., McGartland, L., and

Kaul, K., 1998, Diagnosis of pulmonary tuberculosis using PCR assays on

sputum collected within 24 hours of hospital admission, Am. J. Respir. Crit.

Care Med., 157, 156.

Page 80: Tuberculosis. Pathogenesis and lab diagnosis

57- Catazaro, A., Davidson B. L., Fujiwara, P. I., Goldberger, M. J., Gordin, F.,

Salfinger, M., Sbabaro, J., Schluger, N. W., Sierro, M. F., and Woods, G. L.,

1997, Rapid diagnosis tests for tuberculosis. What is the appropriate use? Am. J.

Respir. Crit. Care Med., 155, 1804.

58- Pfyffer, G. E., 1996, Diagnosis performance of amplified Mycobacterium

tuberculosis direct test with cerebrospinal fluid, other nonrespiratory and

respiratory specimens, J. Clin. Microbial., 34, 834.

59- Hellyer, T. J., Desjardin, L. E., Assaf, M. K., Eisenach, K., Cave, M. D., and

Bates, J. H., 1997, Specificity of IS6110-based amplification assays for

Mycobacterium tuberculosis complex, J. Clin. Microbial., 35, 799.

60 – www.cellestis.com

61- Bowry, S., Chan, C.H. Weiss, H., Katz, S., and Zimmerman, H. J., 1970, Hepatic

involvement in pulmonary tuberculosis: histologic and functional characteristic,

Am. Rev. Resp. Dis., 101, 941.

62- Lewis, J. H. and Zimmerman. H. J., 1993, Tuberculosis of the liver and biliary

tract, in Schlossberg, D., Ed., Tuberculosis, Springer verlag, New York, 199.

63- Maartens, G., Willcox, P. A., and Benatar, S. R., 1990, Miliary tuberculosis: rapid

diagnosis, hematologic abnormalities, and outcome in 109 treated adults, AJM,

89, 291.

64- Prout, S. and Benatar, S. R., 1980, Disseminated tuberculosis: a study of 62 cases,

S. Afr. Med. J., 8, 83.

65- Pettengell, K. E., Larsen, C., Garb, M., Mayet, F. G. H., Simjee, A. E., and Pirie,

D., 1990, Gasrtointestinal tuberculosis in patients with pulmonary tuberculosis,

Q. J. Med., 74, 303.

66- Harrington, P. T., Gutierrez, J. J., Ramirez-Ronda, C. H., Quinones-Soto, R.,

Bermudez. R. H., and Chaffey, J., 1982, Granulomatous hepatitis, Rev. Inf. Dis.,

4, 638.

67- Ratanarapee, S., and Pausawasdi, A., 1991, Tuberculosis of the common bile duct,

HPB Surg., 3, 205.

68- Gooi, H. C. and Smith, J. M., 1978, Tuberculous pericarditis in Birmingham,

Thorax, 33, 94.

69- Lupaktin. H., Braeu, N., Flomenberg, P., and Simberkoff, M. S., 1992,

Tuberculous abscesses in patients with AIDS, Clin, Inf, Dis., 14, 1040.

70- Tandon, P. N., Tuberculous meningitis (cranial and spinal), in Vinken, P. J.,

Bruyn. G. W., and Klawans, H. L., Eds., 1978, Handbook of Clinical

Neurology: Infections of the Nervous System. North Holland Publishing

Company, Amsterdam, 33, 193.

Page 81: Tuberculosis. Pathogenesis and lab diagnosis

��

71- Auerbach. O., 1978, Tuberculous meningitis: correlation of therapeutic results

with the pathogenesis and pathologic changes. Am. Rev. Tuberc., 64, 408.

72- Kasik, J. E., 1993, Central nervous system tuberculosis, in Schlossberg, D., Ed.,

Tuberculosis, Springer Verlag, New York, 129.

73- Molavi, A. and LeFrock, J. L., 1985, Tuberculosis meningitis, Med. Clin. N. Am.,

69, 315.

74- Ogawa, S. K., Smith, M. A., Brennessel, D. J., and Lowy, F. D., 1987,

Tuberculous meningitis in an urban medical center, Medicine, 66, 317.

75- Berenguer, J., Moreno, S., Languna, F., Vicente, T., Adrados, M., Ortega, A.,

Gonzalez-LaHoz, J., and Bouza, E., 1992, Tuberculous meningitis in patients

infected with the human immunodeficiency virus, NEJM, 326, 668.

76- Humphries, M., 1992, The management of Tuberculous meningitis, Thorax, 47,

77.

77- Haas, E. J., Madhavan, T., Qunin, E. L., Cox, F., Fisher, E., and Burch, K., 1977,

Tuberculous meningitis in an urban general hospital, Arch, Int. Med., 137, 1518.

78- Rooney, J. J., Jr., Crocco, J. A., Kramer, S., and Lyons, H. A., 1976, Further

observations on tuberculin reactions in active tuberculosis, Am. J. Med., 60, 17.

79- Dube. M. P., Holtom, P. D., and Larsen, R. A., 1992, Tuberculous meningitis in

patients with and without human immunodeficiency virus infection, AJM, 93,

520.

80- Himman, A. R., 1956, Tuberculous meningitis at Cleveland Metropolitan General

Hospital 1959-1963, Am. Rev. Dis., 9, 670.

81- Kennedy, D. H. and Fallon, R. J., 1979, Tuberculous meningitis, JAMA, 241,

264.

82- Illingworth, R. S., 1979, Miliary and meningeal tuberculosis: difficulties in

diagnosis, Lancet, 271, 646.

83- Stewart, S. M., 1963, Technical methods: the bacteriological diagnosis of

tuberculous meningitis, J. Clin. Pathol., 6, 241.

84- Leonard, J. M. and Des Prez, R. M., 1990, Tuberculous meningitis, Inf. Dis. Clin.

N. Am., 4, 769.

85- De Cock, K. M., Soro, B., Coulibaly, I. M., and Lucas, S. B., 1992, Tuberculosis

and HIV infection in sub-Saharan Africa. JAMA, 268, 1581.

86- Parsons, M., 1988, Tuberculous Meningitis, Oxford University Press, Oxford, U.

K.

Page 82: Tuberculosis. Pathogenesis and lab diagnosis

��

87- Chawla, R. S., Seth, R. K., Raj, B., abd Saini, A. S., 1991, Adenosine deaminase

levels in cerebrospinal fluid in tuberculosis and bacterial meningitis, Tubercle,

72, 190.

88- Daniel, T. M., 1987, New approaches to the rapid diagnosis of tuberculous

meningitis, J. Inf. Dis., 1, 599.

89- Kadival, G. V., Samuel, A. M., Mazarelo, T. B. M. S., and Chaparas, S. D., 1986,

Sensitivity and Specificity of enzyme-linked immunosorbent assay in the

detection of antigen in tuberculous meningitis cerebrospinal fluids, J. Clin.

Microbiol., 23, 901.

90- Kadival, G. V., Samuel, A. M., Mazarelo, T. B. M. S., and Chaparas, S. D., 1987,

Radioimmunoassay for detection of Mycobacterium tuberculosis antigen in

cerebrospinal fluid of patients with tuberculous meningitis, J. Inf. Dis., 155, 608.

91- Cocito, C. G., 1991, Properties of the mycobacterial antigen complex A60 and its

applications to the diagnosis and prognosis of tuberculosis, Chest, 100, 1687.

92- Daniel, T. M., 1990, The rapid diagnosis of tuberculosis: a selective review, J.

Lab. Clin. Med., 116, 277.

93- Shankar, p., Manjunath, N., Mohan, K. K., Prasad, K., Behari, M., Shriniwas, and

Ahuja, G. K., 1991, Rapid diagnosis of tuberculous meningitis by polymerase

chain reaction, lancet, 337, 5.

94- Cameron, D., Ansari, B. M., and Boyce, J. M. H., 1992, Rapid diagnosis of

tuberculous meningitis, J. Inf. Dis., 24, 334.

95- Bouchama, A. Al-Kawi, M. Z., Kanaan, I., Coates, R., Jallu, A., Rahm, B., and

Siqueira, E. B., 1991, Brain biopsy in tuberculoma: thr risks and benefits,

Neurosurgery, 28, 405.

96- http://en.wikipedia.org/

97- http://www.nhsdirect.nhs.uk/

98- Monica Cheesbrough., 2000, District Laboratory Practice in Tropical Countries,

41, 42.

Page 83: Tuberculosis. Pathogenesis and lab diagnosis

��

FIGURES REFERENCES

Figure 1:

http://elementy.ru/images/news/mycobacterium_tuberculosis_3_300.jpg

Figure2:

http://upload.wikimedia.org/wikipedia/commons/thumb/7/71/Mycobacterium_tubercu

losis_Ziehl-Neelsen_stain_02.jpg/800px-Mycobacterium_tuberculosis_Ziehl-

Neelsen_stain_02.jpg

Figure3:

http://wbbt002.biozentrum.uni-

wuerzburg.de/Forschung/Arbeitsgebiete/arbeitsgebiete5be_clip_image002.jpg

Figure4:

http://www.ilustrados.com/publicaciones/multimedia/ma-tub2.jpg

Figure5:

http://staff.vbi.vt.edu/pathport/pathinfo_images/Mycobacterium_tuberculosis/TBInfec

tion_Fig1_StewartPersistentTB.jpg

Figure6:

http://teaching.path.cam.ac.uk/Abnormal/TB_Tuberculosis/ML_Miliary/LU_Lung/A_

TB_ML_LU_12.jpg

Figure7:

http://homepage.usask.ca/~trn186/pth205/tb4.jpg

figure8:

Henrique Couto Teixeria, Clarice Abramo, Martin Emilio Munk., 2007,

Immunological diagnosis of tuberculosis: problems and strategies for success, 323,

334.

Figure9:

My photo

Figure10:

http://www.geologynet.com/micro/XJF200.jpg

Figure11:

http://www.lung.ca/tb/images/full_archive/107_bacillus.jpg

Figure12:

http://education.med.nyu.edu/courses/old/microbiology/courseware/infect-disease/40-

08-MYC.T-LJ.gif

Figure13:

http://images.emedicinehealth.com/images/4453/4453-4482-17621-21204.jpg

Page 84: Tuberculosis. Pathogenesis and lab diagnosis

��

Figure14:

http://www.biomedcentral.com/content/figures/1471-2334-6-154-2.jpg

Figure15:

http://www.pjms.com.pk/issues/julsep06/fig_tab/tb_fig1.gif

figure16, 17, 18:

www.cellestis.com

Figure19:

http://www9.health.gov.au/immhandbook/images/Fig-1-2-3.jpg

Figure20:

http://www.unbc.ca/nlui/wildlife_diseases_bc/avian_tb_liver.jpg

Figure21:

http://www.hku.hk/patho/pracs/CNS/prac1/V.60%20%20%20%20%20TUBERCULO

US%20MENINGITIS.jpg