Al-Balqa' Applied University

Zarka University College

Faculty of Medical Analysis

Allied Medical Science Department

Pulmonary Tuberculosis

Supervised By:

Dr. Waleed Al-Momani

Presented By:

Alaa Abd El-Jaleel Al-Dlahmeh

At Al-Balqa' Applied University

Amman-Jordan

November 2011

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Al-Balqa' Applied University

Zarka University College

Faculty of Medical Analysis

Allied Medical Science Department

Pulmonary Tuberculosis

Supervisor:

Dr. Waleed Al-Momani.

Approved: .

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Dedication

To all persons who love me, support me, stand for me and never doubt about me.........................

To all my big family specially my Father soul, my Mather & sister Asoma ……………..…………

To all my instructors specially Dr. Waleed Al-Momani ………………….

To all my friends who inspired me …………………………

With all my love and respect ……………..

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Abstract

Tuberculosis can affect virtually any organ system in the body and can be devastating if

left untreated. The increasing prevalence of tuberculosis in both immunocompetent and immunocompromised individuals in recent years makes this disease a topic of universal concern. Because tuberculosis demonstrates a variety of clinical and radiologic findings and has a known propensity for dissemination from its primary site, it can mimic numerous other disease entities. Primary pulmonary tuberculosis typically manifests radiologically as parenchymal disease, lymphadenopathy, pleural effusion, miliary disease, or lobar or segmental atelectasis. In postprimary tuberculosis, the earliest radiologic finding is the development of patchy, ill-defined segmental consolidation. Both computed tomography (CT) and magnetic resonance (MR) imaging are helpful in diagnosing tuberculous spondylitis and tuberculous arthritis. CT is especially useful in depicting gastrointestinal and genitourinary tuberculosis. In tuberculosis involving the central nervous system, CT and MR imaging findings vary depending on the stage of disease and the character of the lesion. A high degree of clinical suspicion and familiarity with the various radiologic manifestations of tuberculosis allow early diagnosis and timely initiation of appropriate therapy, thereby reducing patient morbidity.

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Content

7IntroductionChapter 1 Tuberculosis

91.1 Evolution History Of Disease111.2 Causes121.3 Sings & Symptoms131.4 Transmission141.5 Risk Factors151.6 Pathogenesis

Chapter 2 M.Tuberculosis212.1 Morphology & Characteristic 222.2 Cell Wall Structure232.3 Mycobacterium Culture252.4 Other Mycobacteria

Chapter 3 Diagnostic Laboratory Tests3.1 Specimen Collection For Analysis

273.1.1 Sputum Specimens273.1.2 Gastric Aspirates283.1.3 Bronchial Secretions 283.1.4 Urine Specimens

3.2 Acid Fast Bacillus Staining293.2.1 Ziehl-Neelson Acid Fast Staining293.2.2 Auramine 'O' Staining 293.2.3 Auramine-Rhodamine Staining303.2.4 Kinyoun (Fuchsine) Acid Fast Staining313.3 Physical Examination

3.4 Microbiological Studies313.4.1 Sputum Smear & Culture313.4.2 Alternative Sampling323.4.3 PCR 323.4.4 Other323.5 Radiography323.5.1 Chest X-Ray323.5.2 Abreugraphy

3.6 Immunological Test333.6.1 ALS Assay 333.6.2 Tuberculin Skin Test (TST)333.6.2. A Mantoux Skin Test343.6.2.B Heaf Test353.6.2.C BCG Vaccine And Tuberculin Skin Test353.7 Nucleic Acid Amplification Tests (Naat)

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363.8 γ -Interferon Release AssaysChapter 4 Treatment & Prevention

394.1 Treatment 404.2 Multi Drug Resistance Tuberculosis414.3 Epidemiology & Statistics434.4 Prevention & Control444.5 TB control in Jordan454.6 DOTS (Directly Observed Treatment, Short Course)

References

List of figures

Figure (1-1) : Stained M.Tuberculosis 11Figure (1-2) : Sign & Symptoms 12Figure (1-3) : Transmission By Inhalation 13Figure (1-4) : Stages Of Pulmonary Tuberculosis 16Figure (1-5) : Spread Of M.Tuberculosis 17Figure (1-6) : Respiratory System Anatomy 17Figure (2-1) : Serpentine Cord 21Figure (2-2) : M.Tuberculosis With ZN Stain 21Figure (2-3) : M.Tuberculosis Colonies 21Figure (2-4) : SEPTI-CHEK AFB Culture Bottles 23Figure (3-1) : Positive Sputum AFB Smear 28Figure (3-2) : Sputum Smear Stained With Auramine 29Figure (3-3) : ZN Staining Procedure 29Figure (3-4) : Culture For Patient Specimen 31Figure (3-5) : Colorless Of M.Tuberculosis 32Figure (3-7) : Injection Of PPD 33Figure (3-8) : Measuring The Induration Diameter 33

List of Tables

TABLE (1-1) : M.Tuberculosis Species Complex 11TABLE (1-2) : Tuberculosis Infection Vs. Disease 16TABLE (2-1) : Other Mycobacteria Species 25TABLE (3-1) : Characteristics Of Methods For Clinical Mycobacteriology 37

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Introduction

Up until the mid 1980s, there was a steady decline in the prevalence of tuberculosis. Since that time, however, there has been a resurgence of tuberculosis due to the acquired immunodeficiency syndrome (AIDS) epidemic and the increasing number of drug-resistant strains of Mycobacterium tuberculosis. In addition to immunocompromised individuals, other population groups who are at increased risk include minorities, the poor, alcoholics, immigrants from third-world countries, prisoners, the aged, nursing home residents, and the homeless. Although manifestations of tuberculosis are usually limited to the chest, the disease can affect any organ system and in patients infected with human immunodeficiency virus usually involves multiple extrapulmonary sites including the skeleton, genitourinary tract, and central nervous system. Tuberculosis demonstrates a variety of clinical and radiologic features depending on the organ site involved and has a known propensity for dissemination from its primary site. Thus, tuberculosis can mimic a number of other disease entities, and it is important to be familiar with the various radiologic features of tuberculosis to ensure early, accurate diagnosis.

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CHAPTER 1Pulmonary Tuberculosis

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1.1 Evolution History Of Disease

Tubercular decay has been found in the spines of Egyptian mummies. Pictured: Egyptian mummy in the British Museum. Tuberculosis has been present in humans since antiquity. The earliest unambiguous detection of Mycobacterium tuberculosis is in the remains of bison dated 17,000 years before the present However, whether tuberculosis originated in cattle and then transferred to humans, or diverged from a common ancestor, is currently unclear. Skeletal remains show prehistoric humans (4000 BCE) had TB, and tubercular decay has been found in the spines of Egyptian mummies from 3000-2400 BCE. Phthisis is a Greek term for consumption; around 460 BCE, Hippocrates identified phthisis as the most widespread disease of the times involving coughing up blood and fever, which was almost always fatal. Genetic studies suggest that TB was present in The Americas from about the year 100 CE.

Before the Industrial Revolution, tuberculosis may sometimes have been regarded as vampirism. When one member of a family died from it, the other members that were infected would lose their health slowly. People believed that this was caused by the original victim draining the life from the other family members. Furthermore, people who had TB exhibited symptoms similar to what people considered to be vampire traits. People with TB often have symptoms such as red, swollen eyes (which also creates sensitivity to bright light), pale skin and coughing blood, suggesting the idea that the only way for the afflicted to replenish this loss of blood was by sucking blood.

Although it was established that the pulmonary form was associated with 'tubercles' by Dr Richard Morton in 1689, due to the variety of its symptoms, TB was not identified as a single disease until the 1820s and was not named 'tuberculosis' until 1839 by J. L. Schönlein. During the years 1838–1845, Dr. John Croghan, the owner of Mammoth Cave, brought a number of tuberculosis sufferers into the cave in the hope of curing the disease with the constant temperature and purity of the cave air: they died within a year. The first TB sanatorium opened in 1859 in Sokołowsko, Poland by Hermann Brehmer.

Dr. Robert Koch discovered the tuberculosis bacilli.

The bacillus causing tuberculosis, Mycobacterium tuberculosis, was identified and described on March 24, 1882 by Robert Koch. He received the Nobel Prize in physiology or medicine in 1905 for this discovery. Koch did not believe that bovine (cattle) and human tuberculosis were similar, which delayed the recognition of infected milk as a source of infection. Later, this source was eliminated by the pasteurization process. Koch announced a glycerine extract of the tubercle bacilli as a "remedy" for tuberculosis in 1890, calling it 'tuberculin'. It was not effective, but was later adapted as a test for pre-symptomatic tuberculosis.

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The first genuine success in immunizing against tuberculosis was developed from attenuated bovine-strain tuberculosis by Albert Calmette and Camille Guerin in 1906. It was called 'BCG' (Bacillus of Calmette and Guerin). The BCG vaccine was first used on humans in 1921 in France, but it wasn't until after World War II that BCG received widespread acceptance in the USA, Great Britain, and Germany.

Public health campaigns tried to halt the spread of TB.The promotion of Christmas Seals began in Denmark during 1904 as a way to raise money for tuberculosis programs. It expanded to the United States and Canada in 1907-08 to help the National Tuberculosis Association (later called the American Lung Association).In the United States, concern about the spread of tuberculosis played a role in the movement to prohibit public spitting except into spittoons.

In Europe, deaths from TB fell from 500 out of 100,000 in 1850 to 50 out of 100,000 by 1950. Improvements in public health were reducing tuberculosis even before the arrival of antibiotics, although the disease remained a significant threat to public health, such that when the Medical Research Council was formed in Britain in 1913 its initial focus was tuberculosis research.

It was not until 1946 with the development of the antibiotic streptomycin that effective treatment and cure became possible. Prior to the introduction of this drug, the only treatment besides sanatoria were surgical interventions, including the pneumothorax technique—collapsing an infected lung to "rest" it and allow lesions to heal—a technique that was of little benefit and was largely discontinued by the 1950's. The emergence of multidrug-resistant TB has again introduced surgery as part of the treatment for these infections. Here, surgical removal of chest cavities will reduce the number of bacteria in the lungs, as well as increasing the exposure of the remaining bacteria to drugs in the bloodstream, and is therefore thought increase the effectiveness of the chemotherapy.

Hope that the disease could be completely eliminated have been dashed since the rise of drug-resistant strains in the 1980s. For example, tuberculosis cases in Britain, numbering around 50,000 in 1955, had fallen to around 5,500 in 1987, but in 2000 there were over 7,000 confirmed cases. Due to the elimination of public health facilities in New York and the emergence of HIV, there was resurgence in the late 1980s. The number of those failing to complete their course of drugs is high. NY had to cope with more than 20,000 "unnecessary" TB-patients with multidrug-resistant strains (resistant to, at least, both Rifampin and Isoniazid). The resurgence of tuberculosis resulted in the declaration of a global health emergency by the World Health Organization in 1993.

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1.2 Causes

Tuberculosis (TB) is the most common cause of infection-related death worldwide. In 1993, the World Health Organization (WHO) declared TB to be a global public health emergency.

Mycobacterium tuberculosis, along with M. bovis, M. africanum, and M. microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex. Each member of the TB complex is pathogenic, but M. tuberculosis is pathogenic for humans while M. bovis is usually pathogenic for animals and human infected by eating or drinking contaminated, unpasteurized (raw) milk products from areas where M. bovis is present in cattle. Figure (1-1) M.Tuberculosis.

Tubercle bacilli belong to the order Actinomycetales and family Mycobacteriaceae. Mycobacterium tuberculosis is the most common cause of this disease, and it is seen in the image below. Other rare causes include M bovis and M africanum.

The acid-fast characteristic of the mycobacteria is their unique feature. M tuberculosis is an aerobic, non-spore-forming, nonmotile, slow-growing bacillus with a curved and beaded rod-shaped morphology. It is a very hardy bacillus that can survive under adverse environmental conditions. Humans are the only known reservoirs for M tuberculosis.

Mycobacterium tuberculosis complex refers to a genetically closely related group of Mycobacterium species that can cause tuberculosis.

Table (1-1) Mycobacterium tuberculosis complex

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Mycobacterium tuberculosis complex

Species Reservor

M. Tuberculosis Human

M. Bovis Cattels

M. Africanum Monkeys

M. Microti Voles

M.Mungi ____

M.Bovis Bcg ____

M.Canettii ____

M Pinnipedii ____

1.3 Sings & Symptoms

Main symptoms of variants and stages of tuberculosis, with many symptoms overlapping with other variants, while others are more (but not entirely) specific for certain variants. Multiple variants may be present simultaneously.

When tuberculosis becomes active, 75% of cases involve infection in the lungs (pulmonary TB). Symptoms include chest pain, coughing up blood, and a productive, prolonged cough for more than three weeks. Systemic symptoms include:

Cough that lasts 3 weeks or longer, and can bring up blood

Chest pain

Fever

Fatigue

Unintended weight loss

Loss of appetite figure (1-2) sign and symptoms.

Chills

night sweats

pallor

Other symptoms that may occur with this disease:

Breathing difficulty Chest pain

Wheezing

In the other 25% of active cases, the infection moves from the lungs, causing other kinds of TB, collectively denoted extrapulmonary tuberculosis. This occurs more commonly in immunosuppressed persons and young children. Extrapulmonary infection sites include the pleura in tuberculous pleurisy, the central nervous system in meningitis, the lymphatic system in scrofula of the neck, the genitourinary system in urogenital tuberculosis, and the bones and joints in Pott's disease of the spine. When spread to the bones it is also known as "osseous tuberculosis", a form of Osteomyelitis (as a complication of tuberculosis). An

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especially serious form is disseminated TB, more commonly known as miliary tuberculosis. Extrapulmonary TB may co-exist with pulmonary TB.

1.5 Transmission

Respiratory route (M.Tuberculosis)

• Infection requires of inhalation particles small enough to traverse the upper respiratory defenses and deposit deep in the lung (alveoli).

• Large droplets tend to lodge in the more proximal airways and typically do not result in infection.

• TB germs are passed through the air when a person who is sick with TB disease coughs, sings, sneezes, or laughs.

Figure (1-3) transmission by inhalation.

• To become infected with TB germs, a person usually needs to share air space with someone sick with TB disease (e.g., live, work, or play together).

• The amount of time, the environment, and how sick the person is all contribute to whether or not you get infected.

Intestinal route (M. bovis)

• drinking or eating contaminated, unpasteurized (raw) milk or milk products.(uncommon)

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1.4 Risk Factors

Pulmonary tuberculosis (TB) is caused by the bacteria Mycobacterium tuberculosis (M. tuberculosis). You can get TB by breathing in air droplets from a cough or sneeze of an infected person. This is called primary TB.

In the United States, most people will recover from primary TB infection without further evidence of the disease. The infection may stay asleep or inactive (dormant) for years. However, in some people it can reactivate.

Most people who develop symptoms of a TB infection first became infected in the past. However, in some cases, the disease may become active within weeks after the primary infection.

The following people are at higher risk for active TB:

Elderly Infants

People with weakened immune systems, for example due to AIDS, chemotherapy, diabetes, or certain medications

Your risk of contracting TB increases if you:

Are in frequent contact with people who have TB Have poor nutrition

Live in crowded or unsanitary living conditions

The following factors may increase the rate of TB infection in a population:

Increase in HIV infections Increase in number of homeless people (poor environment and nutrition)

The appearance of drug-resistant strains of TB

In the United States, there are approximately 10 cases of TB per 100,000 people. However, rates vary dramatically by area of residence and socioeconomic status.

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1.6 Pathogenesis

About 90% of those infected with Mycobacterium tuberculosis have asymptomatic, latent TB infection (sometimes called LTBI), with only a 10% lifetime chance that a latent infection will progress to TB disease. However, if untreated, the death rate for these active TB cases is more than 50%.

TB infection begins when the mycobacteria reach the pulmonary alveoli, where they invade and replicate within the endosomes of alveolar macrophages. The primary site of infection in the lungs is called the Ghon focus, and is generally located in either the upper part of the lower lobe, or the lower part of the upper lobe. Bacteria are picked up by dendritic cells, which do not allow replication, although these cells can transport the bacilli to local (mediastinal) lymph nodes. Further spread is through the bloodstream to other tissues and organs where secondary TB lesions can develop in other parts of the lung (particularly the apex of the upper lobes), peripheral lymph nodes, kidneys, brain, and bone. All parts of the body can be affected by the disease, though it rarely affects the heart, skeletal muscles, pancreas and thyroid.

Tuberculosis is classified as one of the granulomatous inflammatory conditions. Macrophages, T lymphocytes, B lymphocytes, and fibroblasts are among the cells that aggregate to form granulomas, with lymphocytes surrounding the infected macrophages. The granuloma prevents dissemination of the mycobacteria and provides a local environment for interaction of cells of the immune system. Bacteria inside the granuloma can become dormant, resulting in a latent infection. Another feature of the granulomas of human tuberculosis is the development of abnormal cell death (necrosis) in the center of tubercles. To the naked eye this has the texture of soft white cheese and is termed caseous necrosis.

If TB bacteria gain entry to the bloodstream from an area of damaged tissue they spread through the body and set up many foci of infection, all appearing as tiny white tubercles in the tissues. This severe form of TB disease is most common in infants and the elderly and is called miliary tuberculosis. People with this disseminated TB have a fatality rate near 100% if untreated. However, If treated early, the fatality rate is reduced to about 10%.

In many people the infection waxes and wanes. Tissue destruction and necrosis are balanced by healing and fibrosis. Affected tissue is replaced by scarring and cavities filled with cheese-like white necrotic material. During active disease, some of these cavities are joined to the air passages bronchi and this material can be coughed up. It contains living bacteria and can therefore spread the infection. Treatment with appropriate antibiotics kills bacteria and allows healing to take place. Upon cure, affected areas are eventually replaced by scar tissue.

If untreated, infection with Mycobacterium tuberculosis can cause lobar pneumonia

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Table (1-2) Tuberculosis: Infection vs. Disease

TB Infection TB disease in lungsMTB present MTB presentTuberculin skin test positive Tuberculin skin test positive

Chest X-ray normal Chest X-ray usually reveals lesion

Sputum smears and cultures negative

Sputum smears and cultures positive

No symptoms Symptoms such as cough, fever, weight loss

Not infectious Often infectious before treatment

Not defined as a case of TB Defined as a case of TB

Figure (1-4) Stages of tuberculosis: 1. primary tuberculosis (skin-, x-ray-). 2. Delayed –type hypersensitivity & cell mediated immunity (skin+, x-ray-). 3. Disseminated tuberculosis (skin-, x-ray+). 4. Latent- dormant tuberculosis (skin+, x-ray-). 5. Active tuberculosis (skin+, x-ray+, sputum+).

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Infection Progression

Stage 1

Droplet nuclei are inhaled. One droplet nuclei contains no more than 3 bacilli. Droplet nuclei are so small that they can remain air-borne for extended periods of time. The most effective (infective) droplet nuclei tend to have a diameter of 5 micrometers. Droplet nuclei are generated by during talking coughing and sneezing. Coughing generates about 3000 droplet nuclei. Talking for 5 minutes generates 3000 droplet nuclei but singing generates 3000 droplet nuclei in one minute. Sneezing generates the most droplet nuclei by far, which can spread to individuals up to 10 feet away.

Figure (1-5) Spread of droplet nuclei from one individual to another. After droplet nuclei are inhaled, the bacteria are nonspecifically taken up by alveolar macrophages. However, the macrophages are not activated and are unable to destroy the intracellular organisms.

Figure (1-6) Tuberculosis begins when droplet nuclei reach the alveoli. When a person inhales air that contains droplets most of the larger droplets become lodged in the upper respiratory tract (the nose and throat), where infection is unlikely to develop . However, the smaller droplet nuclei may reach the small air sacs of the lung (the alveoli), where infection begins.

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Stage 2

Begins 7-21 days after initial infection . MTB multiplies virtually unrestricted within unactivated macrophages until the macrophages burst. Other macrophages begin to extravasate from peripheral blood. These macrophages also phagocytose MTB, but they are also unactivated and hence can't destroy the bacteria.

Stage 3

At this stage lymphocytes begin to infiltrate. The lymphocytes, specifically T-cells, recognize processed and presented MTB antigen in context of MHC molecules. This results in T-cell activation and the liberation of cytokines including gamma interferon (IFN). The liberation of IFN causes in the activation of macrophages. These activated macrophages are now capable of destroying MTB.

It is at this stage that the individual becomes tuberculin-positive. This positive tuberculin reaction is the result of the host developing a vigorous cell mediated immune (CMI) response. A CMI response must be mounted to control an MTB infection. An antibody mediated immune (AMI) will not aid in the control of a MTB infection because MTB is intracellular and if extracellular, it is resistant to complement killing due to the high lipid concentration in its cell wall.

Although a CMI response is necessary to control an MTB infection, it is also responsible for much of the pathology associated with tuberculosis. Activated macrophages may release lytic enzymes and reactive intermediates that facilitate the development of immune pathology. Activated macrophages and T-cells also secrete cytokines that may also play a role in the development of immune pathology, including Interleukin 1 ( IL-l), tumor necrosis factor (TNF), and gamma IFN.

It is also at this stage that tubercle formation begins. The center of the tubercle is characterized by "caseation necrosis", meaning it takes on a semi-solid or "cheesy" consistency. MTB cannot multiply within these tubercles because of the low pH and anoxic environment. MTB can, however, persist within these tubercles for extended periods.

Stage 4

Although many activated macrophages can be found surrounding the tubercles, many other macrophages present remain unactivated or poorly activated. MTB uses these macrophages to replicate, and hence, the tubercle grows.

The growing tubercle may invade a bronchus. If this happens, MTB infection can spread to other parts of the lung. Similarly the tubercle may invade an artery or other blood supply line. The hematogenous spread of MTB may result in extrapulmonary tuberculosis otherwise known as milliary tuberculosis. The name "milliary" is derived from the fact that metastasizing tubercles are about the same size as a millet seed, a grain commonly grown in Africa.

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The secondary lesions caused by milliary TB can occur at almost any anatomical location, but usually involve the genitourinary system, bones, joints, lymph nodes and peritoneum. These lesions are of two types:

1. Exudative lesions result from the accumulation of PMN's around MTB. Here the bacteria replicate with virtually no resistance. This situation gives rise to the formation of a "soft tubercle".

2. Productive or granulomatous lesions occur when the host becomes hypersensitive to tuberculoproteins. This situation gives rise to the formation of a "hard tubercle".

Stage 5

For unknown reasons, the caseous centers of the tubercles liquefy. This liquid is very conducive to MTB growth, and the organism begins to rapidly multiply extracellularly. After time, the large antigen load causes the walls of nearby bronchi to become necrotic and rupture. This results in cavity formation. This also allows MTB to spill into other airways and rapidly spread to other parts of the lung.

As stated previously, only a very small percent of MTB infections result in disease, and even a smaller percentage of MTB infections progress to an advanced stage. Usually the host will begin to control the infection at some point. When the primary lesion heals, it becomes fibrous and calcifies. When this happens the lesion is referred to as the Ghon complex. Depending on the size and severity, the Ghon complex may never subside. Typically, the Ghon complex is readily visible upon chest X-ray.

Small metastatic foci containing low numbers of MTB may also calcify. However, in many cases these foci will contain viable organisms. These foci are referred to Simon foci. The Simon foci are also visible upon chest X-ray and are often the site of disease reactivation.

Reactivation of Latent TB to become active infection

TB becomes latent when an infected person’s immune system isn't strong enough to keep the infectious bacteria in check. Presence of the Mycobacterium tuberculosis bacteria causes an immune response in which many types of white blood cells are recruited to sites where the bacteria are growing. They form a walled off lesion, known as a “tubercle” or “granuloma." The bacteria within the tubercle can survive for decades, and conditions leading to a weakened immune response can allow the bacteria to break out of the lesion and reactivate to develop into active TB.

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Chapter 2M.Tuberculosis

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2.1 Morphology & Characteristics

Mycobacterium tuberculosis

"Captain among these Men of Death" (John Bunyon 1660)White DeathWhite PlagueConsumptionTuberculosis...

Mycobacterium tuberculosis is a fairly large nonmotile rod-shaped bacterium distantly related to the Actinomycetes. Many non pathogenic mycobacteria are components of the normal flora of humans, found most often in dry and oily locales. The rods are 2-4 micrometers in length and 0.2-0.5 um in width.

Mycobacterium tuberculosis is an obligate aerobe. For this reason, in the classic case of tuberculosis, MTB complexes are always found in the well-aerated upper lobes of the lungs. The bacterium is a facultative intracellular parasite, usually of macrophages, and has a slow generation time, 15-20 hours, a physiological characteristic that may contribute to its virulence.

Two media are used to grow MTB Middlebrook's medium which is an agar based medium and Lowenstein-Jensen medium which is an egg based medium. MTB colonies are small and buff colored when grown on either medium. Both types of media contain inhibitors to keep contaminants from out-growing MT. It takes 4-6 weeks to get visual colonies on either type of media.

Chains of cells in smears made from in vitro-grown colonies often form distinctive serpentine cords. This observation was first made by Robert Koch who associated cord factor with virulent strains of the bacterium.

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Figure (2-1) Serpentine cord. Figure (2-2) M.Tuberculosis with ZN stain. Figure (2-3) M. tuberculosis Colonies.

MTB is not classified as either Gram-positive or Gram-negative because it does not have the chemical characteristics of either, although the bacteria do contain peptidoglycan (murein) in their cell wall. If a Gram stain is performed on MTB, it stains very weakly Gram-positive or not at all (cells referred to as "ghosts").

Mycobacterium species, along with members of a related genus Nocardia, are classified as acid-fast bacteria due to their impermeability by certain dyes and stains. Despite this, once stained, acid-fast bacteria will retain dyes when heated and treated with acidified organic compounds. One acid-fast staining method for Mycobacterium tuberculosis is the Ziehl-Neelsen stain. When this method is used, the MTB. Smear is fixed, stained with carbol-fuchsin (a pink dye), and decolorized with acid-alcohol. The smear is counterstained with

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methylene-blue or certain other dyes. Acid-fast bacilli appear pink in a contrasting background.

In order to detect Mycobacterium tuberculosis in a sputum sample, an excess of 10,000 organisms per ml of sputum are needed to visualize the bacilli with a 100X microscope objective (1000X mag). One acid-fast bacillus/slide is regarded as "suspicious" of an MTB infection.

2.2 Cell Wall Structure

The cell wall structure of Mycobacterium tuberculosis deserves special attention because it is unique among procaryotes, and it is a major determinant of virulence for the bacterium. The cell wall complex contains peptidoglycan, but otherwise it is composed of complex lipids. Over 60% of the mycobacterial cell wall is lipid. The lipid fraction of MTB's cell wall consists of three major components, mycolic acids, cord factor, and wax-D.

Mycolic acids are unique alpha-branched lipids found in cell walls of Mycobacterium and Corynebacterium. They make up 50% of the dry weight of the mycobacterial cell envelope. Mycolic acids are strong hydrophobic molecules that form a lipid shell around the organism and affect permeability properties at the cell surface. Mycolic Acids are thought to be a significant determinant of virulence in MTB. Probably, they prevent attack of the mycobacteria by cationic proteins, lysozyme, and oxygen radicals in the phagocytic granule. They also protect extracellular mycobacteria from complement deposition in serum.

Cord Factor is responsible for the serpentine cording mentioned above. Cord factor is toxic to mammalian cells and is also an inhibitor of PMN migration. Cord factor is most abundantly produced in virulent strains of MTB.

Wax-D in the cell envelope is the major component of Freund's complete adjuvant (CFA).

2.3 Mycobacterium Culture

Culture of mycobacterium is the definitive method to detect bacilli. It is also more sensitive than examination of the smear. Approximately 10 acid-fast bacilli (AFB) per millimeter of a digested concentrated specimen are sufficient to detect the organisms by culture.

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Another advantage of culture is that it allows specific species identification and testing for recognition of drug susceptibility patterns. However, because M tuberculosis is a slow-growing organism, a period of 6-8 weeks is required for colonies to appear on conventional culture media.

2.3.1 Conventional Growth Techniques

Conventional solid media include the Löwenstein-Jensen medium, which is an egg-based medium, and the Middlebrook 7H10 and the 7H11 media, which are agar-based media. Liquid media (eg, Dubos oleic-albumin media) are also available, and they require incubation in 5-10% carbon dioxide for 3-8 weeks. These media usually have antibacterial antibiotics, which are slightly inhibitory for tubercle bacilli.

2.3.2 Rapid Growth Techniques

Because mycobacteria require 6-8 weeks for isolation from conventional media, automated radiometric culture methods (eg, BACTEC) are increasingly used for the rapid growth of mycobacteria. The methodology uses a liquid Middlebrook 7H12 medium that contains radiometric palmitic acid labeled with radioactive carbon-14 (14 C). Several antimicrobial agents are added to this medium to prevent the growth of nonmycobacterial contaminants. Production of14 CO2 by the metabolizing organisms provides a growth index for the mycobacteria. Growth is generally detected within 9-16 days. Figure (2-4)SEPTI-CHEK AFB Culture Bottle

Another rapid method for isolation of mycobacteria is SEPTICHEK. This nonradiometric approach has a biphasic broth-based system that decreases the mean recovery time versus conventional methods.

Mycobacterial growth indicator tubes (MGITs), which presently are used as a research tool, have round-bottom tubes with oxygen-sensitive sensors at the bottom. MGITs indicate microbial growth and provide a quantitative index of M tuberculosis growth.

2.3.3 Species Identification

M.tuberculosis can be reliably differentiated from other species on the basis of culture characteristics, growth parameters, and other empiric tests. M tuberculosis produces heat-sensitive catalase, reduces nitrates, produces niacin, and grows slowly. Serpentine cording is demonstrated on smears prepared from the BACTEC system.

Addition of p -nitro-acetyl-amino-hydroxy-propiophenone (NAP) inhibits the growth of M tuberculosis complex (including M bovis and M africanum) but does not inhibit growth of other mycobacteria. This provides the basis for the NAP differentiation test.

Chromatographic analysis of mycobacterial cell wall lipids can provide further speciation. The most useful approaches include gas-liquid chromatography and high-performance liquid chromatography (HPLC). The unique mycolic acid pattern associated with the species can be detected by the chromatographic separation of the ester.

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A significant drawback of these chromatographic methods is the requirement of bacterial colonies grown in conventional solid media, a process that takes at least 3 weeks. However, the recent combination of HPLC with fluorescence detection has made the method more sensitive; thus, BACTEC broth culture can be used instead of conventional solid media. This may make the method comparable to the NAP and AccuProbe tests (see Nucleic Acid Probes). The expense of the initial equipment limits the availability of HPLC.

2.3.3. A

Nucleic Acid Probes

Because biochemical methods are time-consuming and laborious, nucleic acid hybridization using molecular probes has become widely accepted. The basic principle is the use of a chemiluminescent, ester-labeled, single-strand DNA probe. A luminometer is used to assess the chemiluminescence.

Commercially available probes, including the AccuProbe technology, help advance identification of the M tuberculosis complex. Sensitivity and specificity approach 100% when at least 100,000 organisms are present.

Positive test results should be reported as M tuberculosis complex, because the probe does not reliably differentiate between M tuberculosis and other members of the complex (eg, M bovis). In addition, final identification to species level is required, because pyrazinamide should not be included in the treatment regimen if the isolate is M bovis.

Niacin production, nitrate reduction, pyrazinamidase, and susceptibility to thiophene-2-carboxylic acid hydrazide can help differentiate between M bovis and M tuberculosis.

2.3.3. B

Nucleic Acid Amplification Tests

Nucleic acid amplification techniques (eg, polymerase chain reaction [PCR]) allows the direct identification of M tuberculosis in clinical specimens, unlike the nucleic acid probes, which require substantial time for bacterial accumulation in broth culture.

The US Food and Drug Administration (FDA) have approved at least 2 tests, the amplified M tuberculosis direct test and the AMPLICOR M tuberculosis test. The amplified M tuberculosis direct test is an isothermal transcription-mediated amplification that targets RNA. The AMPLICLOR test targets the DNA. The most commonly used target sequence for the detection of M tuberculosis has been the insertion sequence IS6110.

Although amplification techniques are promising tools for the rapid diagnosis of tuberculosis (TB), several caveats remain. Contamination of samples by products of previous amplification and the presence of inhibitors in the sample may lead to false-positive or false-negative results.

Although the sensitivity and specificity of the nucleic acid techniques in smear-positive cases exceed 95%, the sensitivity of smear-negative cases varies from 40% to 70%. Thus,

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discordance between the acid-fast smear result and the nucleic acid amplification techniques requires careful clinical appraisal and judgment.

2.4 Other Mycobacteria Species Reservoir Common clinical

manifestation,comment

Species always considered pathogens

Mycobacteria tuberculosis

Humans Pulmonary & disseminated tuberculosis

Mycobacteria lepra Humans Leprosy

Mycobacteria bovis Humans , cattle Tuberculosis-like disease

Species potentially pathogens to humans (moderately )

Mycobacteria avium complex

Soil , water , birds , fowl , cattle , swine ,

environment

Disseminated , Pulmonary; common in AIDS patients

Mycobacteria kansasii Water , cattle Pulmonary , other sites

Species potentially pathogens to humans (very rare )

Mycobacteria africaum Humans , monkeys Pulmonary cultures ; resembles Mycobacteria tuberculosis

Mycobacteria genavense

Humans , pet birds Blood in AIDS patients

Rapid growers

Mycobacteria abscessus Soil , water , animals Pulmonary infections

Mycobacteria chelonae Soil , water , marine life , animals

Cutaneous lesions most common

Table (2-1) other mycobacteria species

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Chapter 3Diagnostic Laboratory Test

3.1 Specimen Collection For Analysis

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The initial step in detection and isolation of the mycobacterium is to obtain appropriate specimens for bacteriologic examination. Examination of sputum, gastric lavage, bronchoalveolar lavage, lung tissue, lymph node tissue, bone marrow, blood, liver, cerebrospinal fluid (CSF), urine, and stool may be useful, depending on the location of the disease.

Decontamination of other microorganisms in the specimens obtained may be performed by the addition of sodium hydroxide, usually in combination with N -acetyl-L -cysteine. Other body fluids (eg, CSF, pleural fluid, peritoneal fluid) can also be centrifuged; the sediment can be stained and evaluated for presence of acid-fast bacilli (AFB). CSF smear results are positive in fewer than 10% of patients in some series. Enhancement of the yield may be possible by staining any clot that may have formed in standing CSF specimens, as well as using the sediment of a centrifuged specimen. Increased yield may also be obtained from cisternal or ventricular fluid.

3.1.1 Sputum Specimens

Sputum specimens may be used in older children, but not in very young children (< 6 y), who usually do not have a cough deep enough to produce sputum for analysis. In those younger than 6 years, gastric aspirates are used.

Nasopharyngeal secretions and saliva are not acceptable. In older children, bronchial secretions may be obtained by the stimulation of cough by an aerosol solution of propylene glycol in 10% sodium chloride (see Bronchial secretions).

3.1.2 Gastric Aspirates

Gastric aspirates are used in lieu of sputum in children younger than 6 years.

Using the correct technique for obtaining the gastric lavage is important because of the scarcity of the organisms in children compared with adults. An early morning sample should be obtained before the child has had a chance to eat or ambulate, because these activities dilute the bronchial secretions accumulated during the night.

Initially, the stomach contents should be aspirated, and then a small amount of sterile water is injected through the orogastric tube. This aspirate should also be added to the specimen.

Because gastric acidity is poorly tolerated by the tubercle bacilli, neutralization of the specimen should be performed immediately with 10% sodium carbonate or 40% anhydrous sodium phosphate. Even with careful attention to detail and meticulous technique, the tubercle bacilli can be detected in only 70% of infants and in 30-40% of children with disease.

3.1.3 Bronchial Secretions

Bronchoalveolar lavage may be used in older children (6 y or older). Bronchial secretions may be obtained by the stimulation of cough by an aerosol solution of propylene glycol in

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10% sodium chloride. This technique may also be used to provide bronchial secretions for detection of tubercle bacilli.

3.1.4 Urine Specimens

Obtain overnight urine specimens in the early morning. Send immediately for analysis, because the tubercle bacilli poorly tolerate the acidic pH of urine.

3.2 Acid Fast Bacillus Staining

Because M tuberculosis is an acid-fast bacilli (AFB), AFB staining provides preliminary confirmation of the diagnosis. Conventional methods include the Ziehl-Neelsen staining method. The Kinyoun stain is modified to make heating unnecessary. Fluorochrome stains, such as auramine and rhodamine, are variations of the traditional stains. The major advantage of these methods is that slides can be screened faster, because the acid-fast material stands out against the dark, nonfluorescent background. However, fluorochrome-positive smears must be confirmed by Ziehl-Neelsen staining.

Staining can also give a quantitative assessment of the number of bacilli being excreted (eg, 1+, 2+, and 3+). This can be of clinical and epidemiologic importance in estimating the infectiousness of the patient and in determining the discontinuation of respiratory isolation. However, for reliably producing a positive result, smears require approximately 10,000 organisms/mL. Therefore, in early stages of the disease or in children in whom the bacilli in the respiratory secretions are sparse, the results may be negative. A single organism on a slide is highly suggestive and warrants further investigation.

A significant drawback of AFB smears is that they cannot be used to differentiate M tuberculosis from other acid-fast organisms such as other mycobacterial organisms or Nocardia species.

Figure (3-1) AFB smear (Note the small, purple, rod-shaped bacilli).

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Figure (3-2) A sputum smear stained with auramine.

3.2.1 Ziehl-Neelson Acid Fast Staining

Acid fast organisms will appear red while non acid fast organisms will stain blue if methylene blue is used as the counterstain or green in the case of brilliant green stain.

Reagents needed...

-Carbol Fuchsin (ZN)-Acid Alcohol Decolouriser

-Methylene Blue 1% or Brilliant Green 1%

Procedure

1. Place the 'fixed' slide on a staining rack and flood copiously with Ziehl-Neelson stain. Apply heat underside of slide for 3 minutes, but do not allow the stain to boil.

2. Wash off surplus stain with distilled water.

3. Destain with acid alcohol until no more colour runs from the smear.

4. Rinse thoroughly with distilled water.

5. Flood slide with methylene blue or brilliant green for 1 or 2 minutes.

6. Rinse thoroughly with distilled water and dry in air.

Examine using high non oil magnification and finally verify under oil immersion.

Figure (3-3) ziehl-neelsen acid fast procedure

3.2.2 Auramine 'O' Staining

Identification of Mycobacteria with auramine 'O' is due to the affinity of the mycolic acid for the fluorchrome which occurs in cell walls. The Mycobacteria are observed as

luminous yellow rods against a dark background. Potassium permanganate can help suppress non-specific fluorescence.

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Slides stained with auramine 'O' may be re-stained later with Ziehl-Neelsen or Kinyoun stain directly, providing that any immersion oil has been removed.

Reagents needed...

Auramine 'O'Fluorescent Decolourising bleach

Potassium Permanganate solution

Procedure

1. Place the 'fixed' smear in a staining rack and flood the slide with auramine 'O' for 15 minutes. Do not allow the surface dry out.

2. Wash off the stain with distilled water.

3. Flood slide with the fluorescent decolouriser for between 30 and 60 seconds.

4. Rinse thoroughly with distilled water.

5. Flood slide with potassium permanganate solution for 2 minutes. Do not allow the surface dry out.

6. Wash thoroughly with distilled water and air dry.

Excitation :

To illuminate the slide, use the same light source as used for fluorescent microscopy. These are usually either powerful mercury or xenon arc-discharge (burner) lamps that contain a combination of dichroic mirrors and filters capable of exciting fluorescent chromophores

and filtering out the excitation light from the viwed image.

Filter combinationsK530 excitation filter with BG 12 barrier

G-365 excitation filter and an LP 420 barrier filterMercury lamps have peaks of intensity at 313, 334, 365, 406, 435, 546, and 578 nanometers

3.2.3 Auramine-Rhodamine Staining

In this method the Mycobacteria appear bright yellow or orange against a greenish background.

Reagents needed...

-Rhodamine-Auramine-Fluorescent Decolourising bleach

-Potassium Permanganate solution

Procedure:

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1. Insert the 'fixed' smear in the staining rack and immerse the slide in rhodamine-auramine for about 15 minutes. Do not allow the surface to dry out.

2. Wash off the stain with distilled water.

3. Flood the slide with fluorescent decolouriser for between 2 and 3 minutes.

4. Wash with distilled water.

5. Flood the slide with potassium permanganate solution for 3 or 4 minutes. Do not allow the surface to dry out.

6. Rinse completely using distilled water and dry in warm air.

3.2.4 Kinyoun (Fuchsin) Acid Fast Staining

Reagents needed...

- Kinyoun Carbol Fuchsin-Acid Alcohol

Procedure:

1. Place the 'fixed' slide on a staining rack and flood it with Kinyoun stain for 2 to 3 minutes.2. Wash off the stain using distilled water.

3. Decolourise the specimen with acid alcohol until no more colour runs from the smear.

4. Rinse thoroughly using distilled water.

5. Counterstain the slide with methylene blue or brilliant green for 1-2 minutes.

6. Rinse thoroughly with distilled water and dry in air.

3.3 Physical Examination

A physical examination is done to assess the patient's general health and find other factors which may affect the TB treatment plan. It cannot be used to confirm or rule out TB. However, certain findings are suggestive of TB. For example, blood in the sputum, significant weight loss and drenching night sweats may be due to TB.

3.4 Microbiological Studies

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Figure (3-4) Doing culture for the patient specimen. Figure (3-5) colorless colony M. tuberculosis.

A definitive diagnosis of tuberculosis can only be made by culturing Mycobacterium tuberculosis organisms from a specimen taken from the patient (most often sputum, but may also include pus, CSF, biopsied tissue, etc.). A diagnosis made other than by culture may only be classified as "probable" or "presumed". For a diagnosis negating the possibility of tuberculosis infection, most protocols require that two separate cultures both test negative.

3.4.1 Sputum Smear & Culture

Sputum smears and cultures should be done for acid-fast bacilli if the patient is producing sputum. The preferred method for this is fluorescence microscopy (auramine-rhodamine staining), which is more sensitive than conventional Ziehl-Neelsen staining. In cases where there is no spontaneous sputum production, a sample can be induced, usually by nebulized inhalation of a saline or saline with bronchodilator solution. A comparative study found that inducing three sputum samples is more sensitive than three gastric washings.

3.4.2 Alternative Sampling

In patients incapable of producing a sputum sample, common alternative sample sources for diagnosing pulmonary tuberculosis include gastric washings, laryngeal swab, bronchoscopy (with bronchoalveolar lavage, bronchial washings, and/or transbronchial biopsy), and fine needle aspiration (transtracheal or transbronchial). In some cases, a more invasive technique is necessary, including tissue biopsy during mediastinoscopy or thoracoscopy.

3.4.3 PCR

Other mycobacteria are also acid-fast. If the smear is positive, PCR or gene probe tests can distinguish M. tuberculosis from other mycobacteria. Even if sputum smear is negative, tuberculosis must be considered and is only excluded after negative cultures.

3.4.4 Other

Many types of cultures are available. Traditionally, cultures have used the Löwenstein-Jensen (LJ), Kirchner, or Middlebrook media (7H9, 7H10, and 7H11). A culture of the AFB can distinguish the various forms of mycobacteria, although results from this may take four to eight weeks for a conclusive answer. New automated systems that are faster include the MB/BacT, BACTEC 9000, and the Mycobacterial Growth Indicator Tube (MGIT). The Microscopic Observation Drug Susceptibility assay culture may be a faster and more accurate method.

3.5 Radiography

3.5.1Chest X-ray

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Tuberculosis creates cavities visible in x-rays like this one in the patient's right upper lobe figure (3-6) chest x-rays.

In active pulmonary TB, infiltrates or consolidations and/or cavities are often seen in the upper lungs with or without mediastinal or hilar lymphadenopathy or pleural effusions ( tuberculous pleurisy). However, lesions may appear anywhere in the lungs. In disseminated TB a pattern of many tiny nodules throughout the lung fields is common - the so called miliary TB. In HIV and other immunosuppressed persons, any abnormality may indicate TB or the chest X-ray may even appear entirely normal. Figure (3-6) chest x-rays

Abnormalities on chest radiographs may be suggestive of, but are never diagnostic of, TB. However, chest radiographs may be used to rule out the possibility of pulmonary TB in a person who has a positive reaction to the tuberculin skin test and no symptoms of disease.

Cavitation or consolidation of the apexes of the upper lobes of the lung may be discernible by a chest x-ray.

3.5.2 Abreugraphy

A variant of the chest X-Ray, abreugraphy (from the name of its inventor, Dr. Manuel Dias de Abreu) was a small radiographic image, also called miniature mass radiography (MMR) or miniature chest radiograph. Though its resolution is limited (it doesn't allow the diagnosis of lung cancer, for example) it is sufficiently accurate for diagnosis of tuberculosis.

Much less expensive than traditional X-Ray, MMR was quickly adopted and extensively utilized in some countries, in the 1950s. For example, in Brazil and in Japan, tuberculosis prevention laws went into effect, obligating ca. 60% of the population to undergo MMR screening.

The procedure went out of favor, as the incidence of tuberculosis dramatically decreased, but is still used in certain situations, such as the screening of prisoners and immigration applicants.

3.6 Immunological Test

3.6.1 ALS Assay

Antibodies from Lymphocyte Secretion or Antibody in Lymphocyte Supernatant or (ALS Assay) is an immunological assay to detect active diseases like tuberculosis, cholera, typhoid etc. Recently, ALS assay nods the scientific community as it is rapidly used for diagnosis of Tuberculosis. The principal is based on the secretion of antibody from in vivo activated plasma B cells found in blood circulation for a short period of time in response to TB-antigens during active TB infection rather than latent TB infection.

3.6.2 Tuberculin Skin Test (TST)

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Two tests are available: the Mantoux & Heaf tests.

3.6.2. A Mantoux Skin Test

Figure (3-7) Injecting of PPD. Figure (3-7) measuring the indurations diameter.

The Mantoux test for TB involves intradermally injecting PPD (Purified Protein Derivative) tuberculin and measuring the size of induration 48-72 hours later.

The Mantoux skin test is used in the United States and is endorsed by the American Thoracic Society and Centers for Disease Control and Prevention (CDC).

3.6.2. B Heaf Test

The Heaf test was used in the United Kingdom until 2005, and is graded on a four point scale. The Mantoux test is now used.

The equivalent Mantoux test positive levels done with 10 TU (0.1 ml 100 TU/ml, 1:1000) are

0–4 mm induration (Heaf 0 to 1) 5–14 mm induration (Heaf 2)

Greater than 15 mm induration (Heaf 3 to 5)

CDC classification of tuberculin reaction

An induration (palpable raised hardened area of skin) of more than 5–15 mm (depending upon the person's risk factors) to 10 Mantoux units is considered a positive result, indicating TB infection.

5 mm or more is positive in

HIV-positive person

Recent contacts of TB case

Persons with nodular or fibrotic changes on CXR consistent with old healed TB

Patients with organ transplants and other immunosuppressed patients

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10 mm or more is positive in

Recent arrivals (less than 5 years) from high-prevalent countries

Injection drug users

Residents and employees of high-risk congregate settings (e.g., prisons, nursing homes, hospitals, homeless shelters, etc.)

Mycobacteriology lab personnel

Persons with clinical conditions that place them at high risk (e.g., diabetes, prolonged corticosteroid therapy, leukemia, end-stage renal disease, chronic malabsorption syndromes, low body weight, etc.)

Children less than 4 years of age, or children and adolescents exposed to adults in high-risk categories

15 mm or more is positive in

Persons with no known risk factors for TB

(Note: Targeted skin testing programs should only be conducted among high-risk groups)

A tuberculin test conversion is defined as an increase of 10 mm or more within a 2-year period, regardless of age.

3.6.2. C BCG Vaccine And Tuberculin Skin Test

There is disagreement on the use of the Mantoux test on people who have been immunized with BCG. The US recommendation is that in administering and interpreting the Mantoux test, previous BCG vaccination should be ignored; the UK recommendation is that interferon-γ tests should be used to help interpret positive tuberculin tests, also, the UK do not recommend serial tuberculin skin testing in people who have had BCG (a key part of the US strategy). In their guidelines on the use of QuantiFERON Gold the US Centers for Disease Control and Prevention state that whereas Quantiferon Gold is not affected by BCG inoculation tuberculin tests can be affected. In general the US approach is likely to result in more false positives and more unnecessary treatment with potentially toxic drugs; the UK approach is as sensitive in theory and should also be more specific, because of the use of interferon-γ tests.

Under the US recommendations, diagnosis and treatment of latent tuberculosis infection (LTBI) is considered for any BCG-vaccinated person whose skin test is 10 mm or greater, if any of these circumstances are present:

Was in contact with another person with infectious TB Was born or has resided in a high TB prevalence country

Is continually exposed to populations where TB prevalence is high.

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3.7 Nucleic Acid Amplification Tests (NAAT)

This is a heterogeneous group of tests that use the polymerase chain reaction (PCR) technique to detect mycobacterial nucleic acid. These test vary in which nucleic acid sequence they detect and vary in their accuracy. The two most common commercially available tests are the amplified mycobacterium tuberculosis direct test (MTD, Gen-Probe) and Amplicor (Roche Diagnostics). In 2007, a systematic review of NAAT by the NHS Health Technology Assessment Programme concluded that "NAAT test accuracy to be far superior when applied to respiratory samples as opposed to other specimens. Although the results were not statistically significant, the AMTD test appears to perform better than other currently available commercial tests.

In a more recent before-after observational study, found that use of the MTD test reduce inappropriate tuberculosis therapy. The study found the accuracy of the MTD test as follows:

Overall

sensitivity 92% specificity 99%

Smear positive patients

sensitivity 99% specificity 98%

Smear negative patients

sensitivity 62% specificity 99%

3.8 Interferon- Release Assaysγ

Interferon-γ (interferon-gamma) release assays (IGRAs) are exciting new developments in TB infection testing. IGRAs are based on the ability of the Mycobacterium tuberculosis antigens for early secretory antigen target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) to stimulate host production of interferon-gamma. Because these antigens are not present in non-tuberculous mycobacteria or in any BCG vaccine variant, these tests can distinguish latent tuberculosis infection (LTBI).

The blood tests QuantiFERON-TB Gold In Tube and T-SPOT.TB use these antigens to detect people with tuberculosis. Lymphocytes from the patient's blood are incubated with the antigens. These tests are called interferon γ tests and are not equivalent. If the patient has been exposed to tuberculosis before, T lymphocytes produce interferon γ in response. The QuantiFERON-TB Gold In Tube uses an ELISA format to detect the whole blood production of interferon γ with great sensitivity (89%).The distinction between the tests is that QuantiFERON-TB Gold quantifies the total amount of interferon γ when whole blood is

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exposed to the antigens(ESAT-6,CFP-10 and TB 7.7(p4)), whereas Guidelines for the use of the FDA approved QuantiFERON-TB Gold were released by the CDC in December 2005. In October 2007, the FDA gave approval of QuantiFERON-TB Gold In Tube for use in the United States.

The enzyme-linked immunospot assay (ELISPOT) is another blood test available in the UK that may replace the skin test for diagnosis. T-SPOT.TB, a type of ELISPOT assay, counts the number of activated T lymphocytes that secrete interferon γ.

For diagnosing latent TB, three systematic reviews of IGRAs concluded the tests noted excellent specificity for the tests to distinguish latent TB from prior vaccination.

According to a study from Korea, where there is a high prevalence of LTBI, QuantiFERON-TB Gold and T-SPOT.TB have good sensitivity but reduced specificity for diagnosing active TB, due to their ability to detect latent TB. In a recently published metaanalysis, with data from both developed and developing countries, QuantiFERON-TB Gold In Tube had a pooled sensitivity for active TB of 81% and specificity of 99.2%, whereas T-SPOT.TB had a pooled sensitivity of 87.5% and specificity of 86.3%. In head-to-head comparisons, the sensitivity of IGRAs surpassed TST. The authors concluded that IGRAs are "superior to the TST for detecting confirmed active TB disease.

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Table (3-1) characteristics of methods for clinical mycobacteriology

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Chapter 4Treatment & Prevention

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4.1 Treatment

A person with a positive skin test, a normal chest X-ray, and no symptoms most likely has only a few TB germs in an inactive state and is not contagious. Nevertheless, treatment with an antibiotic may be recommended for this person to prevent the TB from turning into an active infection. The antibiotic used for this purpose is called isoniazid (INH). If taken for six to 12 months, it will prevent the TB from becoming active in the future. In fact, if a person with a positive skin test does not take INH, there is a 5%-10% lifelong risk that the TB will become active.

Taking isoniazid can be inadvisable (contraindicated) during pregnancy or for those suffering from alcoholism or liver disease. Also, isoniazid can have side effects. The side effects occur infrequently, but a rash can develop, and the individual can feel tired or irritable. Liver damage from isoniazid is a rare occurrence and typically reverses once the drug is stopped. Very rarely, however, especially in older people, the liver damage (INH hepatitis) can even be fatal. It is important therefore, for the doctor to monitor a patient's liver by periodically ordering blood tests called "liver function tests" during the course of INH therapy. Another side effect of INH is a decreased sensation in the extremities referred to as a peripheral neuropathy. This can be avoided by taking vitamin B6 (pyridoxine), and this is often prescribed along with INH.

A person with a positive skin test along with an abnormal chest X-ray and sputum evidencing TB bacteria has active TB and is contagious. As already mentioned, active TB usually is accompanied by symptoms, such as a cough, fever, weight loss, and fatigue.

Active TB is treated with a combination of medications along with isoniazid. Rifampin (Rifadin), ethambutol (Myambutol), and pyrazinamide are the drugs commonly used to treat active TB in conjunction with isoniazid (INH). Four drugs are often taken for the first two months of therapy to help kill any potentially resistant strains of bacteria. Then the number is usually reduced to two drugs for the remainder of the treatment based on drug-sensitivity testing that is usually available by this time in the course. Streptomycin, a drug that is given by injection, may be used as well, particularly when the disease is extensive and/or the patients do not take their oral medications reliably (termed "poor compliance"). Treatment usually lasts for many months and sometimes for years. Successful treatment of TB is dependent largely on the compliance of the patient. Indeed, the failure of a patient to take the medications as prescribed is the most important cause of failure to cure the TB infection. In some locations, the health department demands direct monitoring of patient compliance with therapy.

Surgery on the lungs may be indicated to help cure TB when medication has failed, but in this day and age, surgery for TB is unusual. Treatment with appropriate antibiotics will usually cure the TB. Without treatment, however, tuberculosis can be a lethal infection. Therefore, early diagnosis is important. Those individuals who have been exposed to a person with TB, or suspect that they have been, should be examined by a doctor for signs of TB and screened with a TB skin test.

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4.2 Multi Drug Resistance M. Tuberculosis

Drug-resistant TB (TB that does not respond to drug treatment) has become a very serious problem in recent years in certain populations. For example, INH-resistant TB is seen among patients from Southeast Asia. The presence of INH-like substances in the cough syrups in that part of the world may play a role in causing the INH resistance. Drug-resistant cases are also often seen in prison populations. However, the major reason for the development of resistance is poorly managed TB care. This can result from poor patient compliance, inappropriate dosing or prescribing of medication, poorly formulated medications, and/or an inadequate supply of medication. Multidrug-resistant tuberculosis (MDR-TB) refers to organisms that are resistant to at least two of the first-line drugs, isoniazide (INH) and Rifampin. More recently, extensively (extremely) drug-resistant tuberculosis (XDR-TB) has emerged. These bacteria are also resistant to three or more of the second-line treatment drugs.

Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB. XDR TB is defined as TB which is resistant to isoniazid and rifampin, plus resistant to any fluoroquinolone and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin).

Because XDR TB is resistant to first-line and secondline drugs, patients are left with treatment options that are much less effective.

XDR-TB is seen throughout the world but is most frequently seen in the countries of the former Soviet Union and Asia.

Preventing XDR-TB from spreading is essential. The World Health Organization (WHO) recommends improving basic TB care to prevent emergence of resistance and the development of proper laboratories for detection of resistant cases. When drug-resistant cases are found, prompt, appropriate treatment is required. This will prevent further transmission. Collaboration of HIV and TB care will also help limit the spread of tuberculosis, both sensitive and resistant strains.

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4.3 Epidemiology & Statistics

People with silicosis have an approximately 30-fold greater risk for developing TB. Silica particles irritate the respiratory system, causing immunogenic responses such as phagocytosis, which results in high lymphatic vessel deposits. It is probably this interference and blockage of macrophage function that increases the risk of tuberculosis.

Persons with chronic renal failure and also on hemodialysis have an increased risk.

Persons with diabetes mellitus have a risk for developing active TB that is two to four times greater than persons without diabetes mellitus, and this risk is likely to be greater in persons with insulin-dependent or poorly controlled diabetes.

Other clinical conditions that have been associated with active TB include gastrectomy with attendant weight loss and malabsorption, jejunoileal bypass, renal and cardiac transplantation, carcinoma of the head or neck, and other neoplasms (e.g., lung cancer, lymphoma, and leukemia).

Given that silicosis greatly increases the risk of tuberculosis, more research about the effect of various indoor or outdoor air pollutants on the disease would be necessary. Some possible indoor sources of silica include paint, concrete, and Portland cement. Crystalline silica is found in concrete, masonry, sandstone, rock, paint, and other abrasives. The cutting, breaking, crushing, drilling, grinding, or abrasive blasting of these materials may produce fine silica dust. It can also be in soil, mortar, plaster, and shingles.

Low body weight is associated with risk of tuberculosis as well. A body mass index (BMI) below 18.5 increases the risk by 2 to 3 times. An increase in body weight lowers the risk. People with diabetes mellitus are at increased risk of contracting tuberculosis, and they have a poorer response to treatment, possibly due to poorer drug absorption.

Diabetes increases the risk of TB three-fold. The correlation between diabetes mellitus and TB is concerning for public health because it shows a distinct connection between a contagious disease and a chronic disease. TB is a highly contagious air-born bacteria. Therefore, contracting tuberculosis depends on whether or not a person comes into contact with the bacteria. Diabetics do not have an increased risk of contracting latent tuberculosis but studies have shown that people with diabetes mellitus are more likely to move from a latent form of TB to an active form of TB. This is where the public concern comes from, because when TB is active it is contagious and potentially fatal.

Other conditions that increase risk include the sharing of needles among IV drug users, recent TB infection or a history of inadequately treated TB, chest X-ray suggestive of previous TB, showing fibrotic lesions and nodules, prolonged corticosteroid therapy and other immunosuppressive therapy, compromised immune system (30–40% of people with AIDS worldwide also have TB), hematologic and reticuloendothelial diseases, such as

43

leukemia and Hodgkin's disease, end-stage kidney disease, intestinal bypass, chronic malabsorption syndromes, vitamin D deficiency, and low body weight.

Twin studies in the 1940s showed that susceptibility to TB was heritable. If one of a pair of twins got TB, then the other was more likely to get TB if he was identical than if he was not. These findings were more recently confirmed by a series of studies in South Africa. Specific gene polymorphisms in IL12B have been linked to tuberculosis susceptibility.

Some drugs, including rheumatoid arthritis drugs that work by blocking tumor necrosis factor-alpha (an inflammation-causing cytokine), raise the risk of activating a latent infection due to the importance of this cytokine in the immune defense against TB.

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4.4 TB control in Jordan

The WHO collaborative programme   TUBERCULOSIS CONTROL

Situation analysis

Jordan has a low incidence of tuberculosis. The estimated incidence rate of all TB cases is 7 per 100,000 populations. Every year, 400 people are estimated to develop TB in the country. 30 % of the cases occur among the productive age groups of the community (aged between 15 to 54 years old).

TB control in Jordan has made considerable progress during the last few years. The National TB Programme (NTP) started implementing DOTS in 1998, and achieved the Regional Targets of DOTS ALL OVER in 1998. In 2000, 303 cases of TB were notified in DOTS areas, of which 89 were smear positive new cases. DOTS case detection rate in 2000 is 70%. In 1999, 102 cases of smear positive new cases were detected in DOTS areas, and 92 of them (91%) were successfully treated. More partners are also involved in TB control. Jordanian Anti TB Association is providing support to the national TB programme through financial aid to TB patients, health education and in numerous other activities. Ministry of Defence has started using DOTS in their health services. Jordan also made good progress in the TB Elimination Initiative by lowering the incidence rate of smear positive new cases to 7 per 100,000 populations.

Main achievements

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1.The National TB Programme (NTP) achieved the Regional Targets of DOTS ALL OVER in 1998.

2.DOTS case detection rate in 2000 is 70%.

3.Treatment success rate in 1999 was 91%

4.More partners are also involved in TB control. Jordanian Anti TB Association is providing support to the national TB programme through financial aid to TB patients, health education and in numerous other activities. Ministry of Defence has started using DOTS in their health services. Jordan also made good progress in the TB Elimination Initiative by lowering the incidence rate of smear positive new cases to 7 per 100,000 populations.

Main constraints

1. The need to sustain the quality of DOTS activities.

2. The need to improve the comprehensiveness of DOTS activities to involve other health care providers such as private health sector.

Objectives

Detect at least 70% of the all existing cases of tuberculosis and successfully treat at least 85% of them by 2003

Make steady progress to enroll all detected TB cases in DOTS by 2005.

4.5 Prevention & Control

Control Polices

prompt and effective treatment of patients with active tuberculosis and carefuly follow-up of their contacts with tuberculin tests and x-rays .

Drug treatment of asymptomatic tuberculin-positive Immunization: various living avirulent tubercle bacilli, particularly BCG (bacillus

calmette-guerin,an attenuated bovine organism) used to induce a certain amount of resistance in those heavily exposed to infection

The eradication of tuberculosis in cattle and the pasteurization of milk have greatly reduced M.bovis infections.

Precotions Polices

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Avoid getting active TB.

Prevent latent TB from becoming active.

A TB vaccine (bacille Calmette-Guerin, or BCG) is used in many countries to prevent TB.

Prevent inject illegal drugs.

Do not spend long periods of time in stuffy, enclosed rooms with anyone who has active TB until that person has been treated for at least 2 weeks.

Use protective measures, such as face masks, if you work in a facility that cares for people who have untreated TB.

4.6 DOTS (Directly Observed Treatment, Short Course)

The WHO-recommended Directly Observed Treatment, Short Course (DOTS) strategy was launched formally as Revised National TB Control programme in India in 1997 after pilot testing from 1993-1996. Since then DOTS has been widely advocated and successfully applied.

A DOT is the most effective strategy available for controlling TB.

The five key components of DOTS are

i. Political commitment to control TB; ii. Case detection by sputum smear microscopy examination among symptomatic

patients;

iii. Patients are given anti- TB drugs under the direct observation of the health care provider/community DOT provider.

iv. Regular, uninterrupted supply of anti-TB drugs.

v. Systematic recording and reporting system that allows assessment of treatment results of each and every patient and of whole TB control programme.

The patient is the VIP of the Programme and responsibility of ensuring regular and complete treatment of the patient lies with the health system.

In 2006, the new stop TB strategy was recommended internationally by WHO. The components of the new stop TB strategy are the following:

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1. Pursue High quality DOTS expansion and enhancement 2. Address TB/HIV, MDR-TB and other challenges

3. Contribute to health system strengthening

4. Engage all health care providers

5. Empower people with TB, and communities

6. Enable and promote research

References

1. G.Brooks, K.Carrroll, J.Butel, S.Melnicks (Medical Microbiology 24th Edition) Jawetz, Melnicks Dahlberg's (2010).

2. Landau, Elaine. Tuberculosis. New York: F. Watts, 1995http://www.healthofchildren.com/T/Tuberculosis.

3. Tuberculosis, NICE Clinical Guideline (March 2011); Clinical diagnosis and management of tuberculosis, and measures for its prevention and control

4. The Stop TB Strategy: building on and enhancing DOTS to meet the TB- related Millennium Development GoalsWHO. Geneva, World Health Organization, 2006b (WHO/HTM/STB/2006.37).

5. Global burden of tuberculosis : estimated incidence, prevalence and mortality by countryDye C et al. Global burden of tuberculosis: estimated incidence,

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prevalence and mortality by country. Journal of the American Medical Association, 1999.

6. Global tuberculosis control: surveillance, planning, financing. WHO report 2008 . Improving outcomes in urological cancers, NICE Cancer Service Guidance (2002)

7. Immunisation against infectious disease - ' The Green Book ' , Dept of Health (various dates)Colditz GA, Brewer TF, Berkey CS, et al; Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature.

8. Aronson NE, Santosham M, Comstock GW, et al; Long-term efficacy of BCG vaccine in American Indians and Alaska Natives: A JAMA. 2004 May

9. Chan ED, Iseman MD ; Current medical treatment for tuberculosis. BMJ. 2002 Nov .

10.Stedwell RE, Allen KM, Binder LS ; Hypokalemic paralyses: a review of the etiologies, pathophysiology, presentation, and therapy. Am J Emerg Med.

11.Mitropoulos DN ; Novel insights into the mechanism of action of intravesical immunomodulators.

12.Davis SD, Yankelevitz DF, Williams T, et al. Pulmonary tuberculosis in immunocompromised hosts: epidemiological, clinical, and radiological assessment. Semin Roentgenol 1993.

13. Floyd K, Blanc L, Raviglione M, Lee JW. Resources required for global tuberculosis control. Science 2002

The End

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