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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


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 30
Examination
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
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
5- Duration of Treatment 64
6- General Procedure that should be followed during 64
Treatment
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
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.
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)
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


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
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)
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
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)
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)
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.
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.
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 Yes Minimal
involvement
Cavity Rare Frequent
formation
Tuberculin Negative (initially) Positive
reactivity
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.
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.
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).
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)
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)
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
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)
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
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)
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
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)
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.


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.
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.
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.
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.
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.


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


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.
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)
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


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
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
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
• 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
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
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


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
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.
*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.
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.
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.
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.
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 Volume of Green


Conjugate 100X Diluents
Concentrate
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.
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.
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.
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.


4In 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.
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.
• 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)
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.
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.
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


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.
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.
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.
- 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.
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)
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)
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)
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,
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.
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)
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)
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.
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