Tuberculosis. Pathogenesis And Lab Diagnosis

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

Contents

Introduction

5

Mycobacterium Tuberculosis Morphology of Mycobacterium Tuberculosis Virulence Factors of Mycobacterium Tuberculosis 1- Cord Factor(s) 2- Mycobacterial Sulfolipids 3- Mycoside Transmission Of Mycobacterium Tuberculosis

8 11 11 11 12 13

Pathogenesis Pathogenesis of Tuberculosis 1- Primary Tuberculosis 2- Post-primary Tuberculosis 3- Immunocompromised individuals Symptoms Of Tuberculosis Immunologic response Immunologic Response Against Tuberculosis Diagnosis Of Tuberculosis Diagnosis Of Tuberculosis 1- Sputum Examination 1-1- Preparation, Staining and Microscopic Examination 2- Culture Method 2-1- Preparation of Specimen 2-2- Incubation Conditions 2-3- Automated System 2-4- Identification 3- X-ray 4- Tuberculin Test 4-1- Description 4-2- Preparation 4-3- Aftercare 4-4- Risks

15 15 18 20 21 23

30 30 30 37 37 37 38 38 39 40 40 40 40 41

4-5- Normal results 4-6- Abnormal Results 5- Polymerase Chain Reaction New Diagnostic Method 1- Intended Used 2- Summary and Explanation of The Test 3- Principle of The Assay 4- Reagent and Storage 4-1- Peptide and Control Antigens 4-2- ELISA Component 4-3- Storage Instructions 5- Specimen Collection and Handling 6- Directions of Use 6-1- Stage One 6-1- Stage Two 7- Interpretation of Results 8- Warnings and Precaution 8-1- Warnings 8-3- Precaution

41 42 43 46 46 46 47 48 48 48 48 49 49 49 51 55 56 56 56

BCG Vaccine BCG Vaccine of Tuberculosis 1- BCG Vaccine 2- Storing and Validity 3- Indication 4- Complication 5- BCG Vaccine and Other Vaccines 6- Administration and Dosage 7- Site

59 59 59 59 60 60 60 61

Treatment Treatment of Tuberculosis 1- General Roles of Tuberculosis Treatment 2- Directly Observed Treatment, Short Course (DOTS) 3- Phases of Treatment 4- Hospitalization

63 63 63 63 64

5- Duration of Treatment 6- General Procedure that should be followed during Treatment 7- Categories of Treatment 8-1- CAT 1 8-2- CAT 2 8-3- CAT 3 8-4- CAT 4

64 64 65 65 65 66 66 67

Extrapulmonary Tuberculosis 1- Hepatic 1-1- Clinical 1-2- Diagnosis 2- Meninges 2-1- Clinical 2-2- Diagnosis

69 69 70 71 71 72

Conclusion

72

References Figures References

76 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 highrisk 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 cellmediated type, which, if mediated by Th1 T helper cells, leads to protective immunity, but the presence of Th2 cells facilitates tissuedestroying 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 postprimary 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 postprimary 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 tuberculosis in non-compromised patients Characteristic Primary Local lesion Small Lymphatic Yes involvement Cavity Rare formation Tuberculin Negative (initially) reactivity Infectivity Uncommon Site Any part of lung Local spread Uncommon

and

post-primary

Post-primary Large Minimal Frequent Positive Usual Apical region 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 nonimmunocompromised 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 CD8+ T cells. (22)

can specifically stimulate

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 proinflammatory 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 IL27, 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 INFproduction 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 IL2 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 CalmetteGuerin (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 preemployment 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 nontuberculosis 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 paraffinembedded 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 EnzymeLinked 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 Tcells 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 10 minutes (add 1 to 1.5 Blood culture set up: * 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 2. ESAT-6 Peptides 3. CFP–10 Peptides 4. Mitogen Control

1 x 6mL 1 x 6mL 1 x 6mL 1 x 6mL

4-2 ELISA Components 1. Microplate strips 2. Human IFN- Standard, lyophilised 3. Green Diluent 4. Conjugate 100X Concentrate, lyophilised 5. Wash Buffer 20X Concentrate 6. Enzyme Substrate Solution 7. Enzyme Stopping Solution

24 x 8 well 1 x vial 1 x 30mL 1 x 0.3mL 1 x 100mL 1 x 30mL 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 rollerrocker 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 STANDARDS

PROCEDUREFOR

DUPLICATE

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

2 3 4 5 6 7 8 9 10 11 12

Volume Of Conjugate 100X Concentrate 10 ul 15 ul 20 ul 25 ul 30 ul 35 ul 40 ul 45 ul 50 ul 55 ul 60 ul

Volume of Green Diluents 1.0 ml 1.5 ml 2.0 ml 2.5 ml 3.0 ml 3.5 ml 4.0 ml 4.5 ml 5.0 ml 5.5 ml 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:

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.

1

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.

2

3

Refer to Section 9 for possible causes.

In clinical studies, less than 0.25% of subjects had IFN- levels of > 8.0 IU/mL for the Nil Control.

4

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: falsenegative 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 HIVpositive 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) Enzymelinked 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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FIGURES REFERENCES Figure 1: http://elementy.ru/images/news/mycobacterium_tuberculosis_3_300.jpg Figure2: http://upload.wikimedia.org/wikipedia/commons/thumb/7/71/Mycobacterium_tubercu losis_Ziehl-Neelsen_stain_02.jpg/800px-Mycobacterium_tuberculosis_ZiehlNeelsen_stain_02.jpg Figure3: http://wbbt002.biozentrum.uniwuerzburg.de/Forschung/Arbeitsgebiete/arbeitsgebiete5be_clip_image002.jpg Figure4: http://www.ilustrados.com/publicaciones/multimedia/ma-tub2.jpg Figure5: http://staff.vbi.vt.edu/pathport/pathinfo_images/Mycobacterium_tuberculosis/TBInfec tion_Fig1_StewartPersistentTB.jpg Figure6: http://teaching.path.cam.ac.uk/Abnormal/TB_Tuberculosis/ML_Miliary/LU_Lung/A_ TB_ML_LU_12.jpg Figure7: http://homepage.usask.ca/~trn186/pth205/tb4.jpg figure8: Henrique Couto Teixeria, Clarice Abramo, Martin Emilio Munk., 2007, Immunological diagnosis of tuberculosis: problems and strategies for success, 323, 334. Figure9: My photo Figure10: http://www.geologynet.com/micro/XJF200.jpg Figure11: http://www.lung.ca/tb/images/full_archive/107_bacillus.jpg Figure12: http://education.med.nyu.edu/courses/old/microbiology/courseware/infect-disease/4008-MYC.T-LJ.gif Figure13: http://images.emedicinehealth.com/images/4453/4453-4482-17621-21204.jpg

Figure14: http://www.biomedcentral.com/content/figures/1471-2334-6-154-2.jpg Figure15: http://www.pjms.com.pk/issues/julsep06/fig_tab/tb_fig1.gif figure16, 17, 18: www.cellestis.com Figure19: http://www9.health.gov.au/immhandbook/images/Fig-1-2-3.jpg Figure20: http://www.unbc.ca/nlui/wildlife_diseases_bc/avian_tb_liver.jpg Figure21: http://www.hku.hk/patho/pracs/CNS/prac1/V.60%20%20%20%20%20TUBERCULO US%20MENINGITIS.jpg