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pulmonary Tuberculosis

  1. 1. Al-Balqa Applied University Zarka University CollegeFaculty of Medical AnalysisAllied Medical Science DepartmentPulmonary Tuberculosis :Supervised By Dr. Waleed Al-Momani :Presented By Alaa Abd El-Jaleel Al-Dlahmeh At Al-Balqa Applied University Amman-Jordan 1
  2. 2. November 2011 Al-Balqa Applied University Zarka University College Faculty of Medical AnalysisAllied Medical Science Department Pulmonary Tuberculosis Supervisor: Dr. Waleed Al- Momani. 2
  3. 3. Approved: .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 …………….. 3
  4. 4. Abstract T uberculosis can affect virtually any organ system in the body and can be devastating ifleft untreated. The increasing prevalence of tuberculosis in both immunocompetent andimmunocompromised individuals in recent years makes this disease a topic of universalconcern. Because tuberculosis demonstrates a variety of clinical and radiologic findings andhas a known propensity for dissemination from its primary site, it can mimic numerousother disease entities. Primary pulmonary tuberculosis typically manifests radiologically asparenchymal disease, lymphadenopathy, pleural effusion, miliary disease, or lobar orsegmental atelectasis. In postprimary tuberculosis, the earliest radiologic finding is thedevelopment of patchy, ill-defined segmental consolidation. Both computed tomography(CT) and magnetic resonance (MR) imaging are helpful in diagnosing tuberculous spondylitisand tuberculous arthritis. CT is especially useful in depicting gastrointestinal andgenitourinary tuberculosis. In tuberculosis involving the central nervous system, CT and MRimaging findings vary depending on the stage of disease and the character of the lesion. Ahigh degree of clinical suspicion and familiarity with the various radiologic manifestations oftuberculosis allow early diagnosis and timely initiation of appropriate therapy, therebyreducing patient morbidity. 4
  5. 5. ContentIntroduction ٧ Chapter 1 TuberculosisEvolution History Of Disease 1.1 ٩Causes 1.2 ١١Sings & Symptoms 1.3 ١٢Transmission 1.4 ١٣Risk Factors 1.5 ١٤Pathogenesis 1.6 ١٥Chapter 2 M.Tuberculosis Morphology & Characteristic 2.1 ٢١Cell Wall Structure 2.2 ٢٢Mycobacterium Culture 2.3 ٢٣Other Mycobacteria 2.4 ٢٥Chapter 3 Diagnostic Laboratory TestsSpecimen Collection For Analysis 3.1Sputum Specimens 3.1.1 ٢٧3.1.2 Gastric Aspirates ٢٧Bronchial Secretions 3.1.3 ٢٨ Urine Specimens 3.1.4 ٢٨Acid Fast Bacillus Staining 3.2Ziehl-Neelson Acid Fast Staining 3.2.1 ٢٩ Auramine O Staining 3.2.2 ٢٩Auramine-Rhodamine Staining 3.2.3 ٢٩Kinyoun (Fuchsine) Acid Fast Staining 3.2.4 ٣٠Physical Examination 3.3 ٣١Microbiological Studies 3.4Sputum Smear & Culture 3.4.1 ٣١3.4.2 Alternative Sampling 313.4.3 PCR 323.4.4 Other 32 5
  6. 6. 3.5 Radiography 323.5.1 Chest X-Ray 323.5.2 Abreugraphy 323.6 Immunological Test3.6.1 ALS Assay 333.6.2 Tuberculin Skin Test (TST) 333.6.2. A Mantoux Skin Test 333.6.2.B Heaf Test 343.6.2.C BCG Vaccine And Tuberculin Skin Test 35(Nucleic Acid Amplification Tests (Naat 3.7 35γ -Interferon Release Assays 3.8 36Chapter 4 Treatment & Prevention Treatment 4.1 39Multi Drug Resistance Tuberculosis 4.2 40Epidemiology & Statistics 4.3 41Prevention & Control 4.4 43TB control in Jordan 4.5 44(DOTS (Directly Observed Treatment, Short Course 4.6 45 References List of figures Figure (1-1) : Stained M.Tuberculosis 11 Figure (1-2) : Sign & Symptoms 12 Figure (1-3) : Transmission By Inhalation 13 Figure (1-4) : Stages Of Pulmonary Tuberculosis 16 Figure (1-5) : Spread Of M.Tuberculosis 17 Figure (1-6) : Respiratory System Anatomy 17 Figure (2-1) : Serpentine Cord 21 Figure (2-2) : M.Tuberculosis With ZN Stain 21 Figure (2-3) : M.Tuberculosis Colonies 21 Figure (2-4) : SEPTI-CHEK AFB Culture Bottles 23 Figure (3-1) : Positive Sputum AFB Smear 28 Figure (3-2) : Sputum Smear Stained With Auramine 29 Figure (3-3) : ZN Staining Procedure 29 Figure (3-4) : Culture For Patient Specimen 31 Figure (3-5) : Colorless Of M.Tuberculosis 32 Figure (3-7) : Injection Of PPD 33 Figure (3-8) : Measuring The Induration Diameter 33 6
  7. 7. List of TablesTABLE (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 37Introduction 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 acquiredimmunodeficiency syndrome (AIDS) epidemic and the increasing number of drug-resistantstrains 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, andthe homeless.Although manifestations of tuberculosis are usually limited to the chest, the disease canaffect any organ system and in patients infected with human immunodeficiency virususually involves multiple extrapulmonary sites including the skeleton, genitourinary tract,and central nervous system.Tuberculosis demonstrates a variety of clinical and radiologic features depending on theorgan 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 befamiliar with the various radiologic features of tuberculosis to ensure early, accuratediagnosis. 7
  8. 8. CHAPTER 1Pulmonary Tuberculosis 8
  9. 9. 1.1 Evolution History Of Disease Tubercular decay has been found in the spines of Egyptianmummies. Pictured: Egyptian mummy in the British Museum.Tuberculosis has been present in humans since antiquity. The earliestunambiguous detection of Mycobacterium tuberculosis is in theremains of bison dated 17,000 years before the present However,whether tuberculosis originated in cattle and then transferred to humans, or diverged froma common ancestor, is currently unclear. Skeletal remains show prehistoric humans (4000BCE) had TB, and tubercular decay has been found in the spines of Egyptian mummies from3000-2400 BCE. Phthisis is a Greek term for consumption; around 460 BCE, Hippocratesidentified phthisis as the most widespread disease of the times involving coughing up bloodand fever, which was almost always fatal. Genetic studies suggest that TB was present inThe Americas from about the year 100 CE.Before the Industrial Revolution, tuberculosis may sometimes have been regarded asvampirism. When one member of a family died from it, the other members that wereinfected would lose their health slowly. People believed that this was caused by the originalvictim draining the life from the other family members. Furthermore, people who had TBexhibited symptoms similar to what people considered to be vampire traits. People with TBoften have symptoms such as red, swollen eyes (which also creates sensitivity to brightlight), pale skin and coughing blood, suggesting the idea that the only way for the afflictedto replenish this loss of blood was by sucking blood.Although it was established that the pulmonary form was associated with tubercles by DrRichard Morton in 1689, due to the variety of its symptoms, TB was not identified as a singledisease 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 anumber of tuberculosis sufferers into the cave in the hope of curing the disease with theconstant temperature and purity of the cave air: they died within a year. The first TBsanatorium opened in 1859 in Sokołowsko, Poland by Hermann Brehmer.Dr. Robert Koch discovered the tuberculosis bacilli. 9
  10. 10. The bacillus causing tuberculosis, Mycobacterium tuberculosis, was identified and describedon March 24, 1882 by Robert Koch. He received the Nobel Prize in physiology or medicine in1905 for this discovery. Koch did not believe that bovine (cattle) and human tuberculosiswere 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 glycerineextract of the tubercle bacilli as a "remedy" for tuberculosis in 1890, calling it tuberculin. Itwas not effective, but was later adapted as a test for pre-symptomatic tuberculosis.The first genuine success in immunizing against tuberculosis was developed fromattenuated bovine-strain tuberculosis by Albert Calmette and Camille Guerin in 1906. It wascalled BCG (Bacillus of Calmette and Guerin). The BCG vaccine was first used on humans in1921 in France, but it wasnt until after World War II that BCG received widespreadacceptance in the USA, Great Britain, and Germany.Public health campaigns tried to halt the spread of TB.The promotion ofChristmas Seals began in Denmark during 1904 as a way to raise money fortuberculosis programs. It expanded to the United States and Canada in1907-08 to help the National Tuberculosis Association (later called theAmerican Lung Association).In the United States, concern about the spread oftuberculosis played a role in the movement to prohibit public spitting exceptinto spittoons.In Europe, deaths from TB fell from 500 out of 100,000 in 1850 to 50 out of 100,000 by1950. Improvements in public health were reducing tuberculosis even before the arrival ofantibiotics, although the disease remained a significant threat to public health, such thatwhen the Medical Research Council was formed in Britain in 1913 its initial focus wastuberculosis research. It was not until 1946 with the development of the antibiotic streptomycin that effectivetreatment and cure became possible. Prior to the introduction of this drug, the onlytreatment besides sanatoria were surgical interventions, including the pneumothoraxtechnique—collapsing an infected lung to "rest" it and allow lesions to heal—a techniquethat was of little benefit and was largely discontinued by the 1950s. The emergence ofmultidrug-resistant TB has again introduced surgery as part of the treatment for theseinfections. Here, surgical removal of chest cavities will reduce the number of bacteria in thelungs, as well as increasing the exposure of the remaining bacteria to drugs in thebloodstream, and is therefore thought increase the effectiveness of the chemotherapy.Hope that the disease could be completely eliminated have been dashed since the rise ofdrug-resistant strains in the 1980s. For example, tuberculosis cases in Britain, numberingaround 50,000 in 1955, had fallen to around 5,500 in 1987, but in 2000 there were over7,000 confirmed cases. Due to the elimination of public health facilities in New York and theemergence of HIV, there was resurgence in the late 1980s. The number of those failing tocomplete 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, bothRifampin and Isoniazid). The resurgence of tuberculosis resulted in the declaration of aglobal health emergency by the World Health Organization in 1993. 10
  11. 11. 1.2 Causes Tuberculosis (TB) is the most common cause ofinfection-related death worldwide. In 1993, the WorldHealth Organization (WHO) declared TB to be a globalpublic health emergency.Mycobacterium tuberculosis, along with M. bovis, M.africanum, and M. microti all cause the disease known astuberculosis (TB) and are members of the tuberculosisspecies complex. Each member of the TB complex ispathogenic, but M. tuberculosis is pathogenic for humanswhile M. bovis is usually pathogenic for animals andhuman infected by eating or drinking contaminated,unpasteurized (raw) milk products from areas where M. bovis ispresent 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 theimage below. Other rare causes include M bovis and M africanum. The acid-fast characteristic of the mycobacteria is their unique feature. M tuberculosis is anaerobic, non-spore-forming, nonmotile, slow-growing bacillus with a curved and beadedrod-shaped morphology. It is a very hardy bacillus that can survive under adverseenvironmental conditions. Humans are the only known reservoirs for M tuberculosis.Mycobacterium tuberculosis complex refers to a genetically closely related group ofMycobacterium species that can cause tuberculosis. Mycobacterium tuberculosis complex Species Reservor M. Tuberculosis Human M. Bovis Cattels M. Africanum Monkeys M. Microti Voles M.Mungi ____ M.Bovis Bcg ____ M.Canettii 11 ____ M Pinnipedii ____
  12. 12. Table (1-1) Mycobacterium tuberculosis complex1.3 Sings & Symptoms Main symptoms of variants and stages oftuberculosis, with many symptoms overlapping withother variants, while others are more (but notentirely) specific for certain variants. Multiplevariants may be present simultaneously.When tuberculosis becomes active, 75% of casesinvolve infection in the lungs (pulmonary TB).Symptoms include chest pain, coughing up blood,and a productive, prolonged cough for more thanthree 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 • pallorOther symptoms that may occur with this disease: • Breathing difficulty • Chest pain • WheezingIn the other 25% of active cases, the infection moves from the lungs, causing other kinds ofTB, collectively denoted extrapulmonary tuberculosis. This occurs more commonly inimmunosuppressed persons and young children. Extrapulmonary infection sites include thepleura in tuberculous pleurisy, the central nervous system in meningitis, the lymphaticsystem in scrofula of the neck, the genitourinary system in urogenital tuberculosis, and thebones and joints in Potts disease of the spine. When spread to the bones it is also known as 12
  13. 13. "osseous tuberculosis", a form of Osteomyelitis (as a complication of tuberculosis). Anespecially 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) 13
  14. 14. 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 aninfected person. This is called primary TB.In the United States, most people will recover from primary TB infection without furtherevidence 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 primaryinfection.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 medicationsYour 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 conditionsThe 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 TBIn 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. 14
  15. 15. 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 latentinfection will progress to TB disease. However, if untreated, the death rate for these activeTB cases is more than 50%.TB infection begins when the mycobacteria reach the pulmonary alveoli, where they invadeand replicate within the endosomes of alveolar macrophages. The primary site of infectionin the lungs is called the Ghon focus, and is generally located in either the upper part of thelower 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 andorgans where secondary TB lesions can develop in other parts of the lung (particularly theapex of the upper lobes), peripheral lymph nodes, kidneys, brain, and bone. All parts of thebody 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 thataggregate to form granulomas, with lymphocytes surrounding the infected macrophages.The granuloma prevents dissemination of the mycobacteria and provides a localenvironment for interaction of cells of the immune system. Bacteria inside the granulomacan become dormant, resulting in a latent infection. Another feature of the granulomas ofhuman tuberculosis is the development of abnormal cell death (necrosis) in the center oftubercles. To the naked eye this has the texture of soft white cheese and is termed caseousnecrosis.If TB bacteria gain entry to the bloodstream from an area of damaged tissue they spreadthrough the body and set up many foci of infection, all appearing as tiny white tubercles inthe tissues. This severe form of TB disease is most common in infants and the elderly and iscalled 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 arebalanced by healing and fibrosis. Affected tissue is replaced by scarring and cavities filledwith cheese-like white necrotic material. During active disease, some of these cavities arejoined to the air passages bronchi and this material can be coughed up. It contains livingbacteria and can therefore spread the infection. Treatment with appropriate antibiotics killsbacteria and allows healing to take place. Upon cure, affected areas are eventually replacedby scar tissue.If untreated, infection with Mycobacterium tuberculosis can cause lobar pneumonia 15
  16. 16. Table (1-2) Tuberculosis: Infection vs. Disease TB Infection TB disease in lungs MTB present MTB present Tuberculin skin test positive Tuberculin skin test positive Chest X-ray normal Chest X-ray usually reveals lesion Sputum smears and cultures Sputum smears and cultures negative 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 TBFigure (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- dormanttuberculosis (skin+, x-ray-). 5. Active tuberculosis (skin+, x-ray+, sputum+). 16
  17. 17. Infection ProgressionStage 1Droplet nuclei are inhaled. One droplet nuclei contains no more than 3 bacilli. Dropletnuclei are so small that they can remain air-borne for extended periods of time. The mosteffective (infective) droplet nuclei tend to have a diameter of 5 micrometers. Droplet nucleiare generated by during talking coughing and sneezing. Coughing generates about 3000droplet nuclei. Talking for 5 minutes generates 3000 droplet nuclei but singing generates3000 droplet nuclei in one minute. Sneezing generates the most droplet nuclei by far, whichcan 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, thebacteria are nonspecifically taken up by alveolar macrophages. However, the macrophages are notactivated and are unable to destroy the intracellular organisms.Figure (1-6) Tuberculosis begins when droplet nuclei reach the alveoli. When a person inhales air thatcontains droplets most of the larger droplets become lodged in the upper respiratory tract (the nose andthroat), where infection is unlikely to develop . However, the smaller droplet nuclei may reach the small airsacs of the lung (the alveoli), where infection begins. 17
  18. 18. Stage 2Begins 7-21 days after initial infection . MTB multiplies virtually unrestricted withinunactivated macrophages until the macrophages burst. Other macrophages begin toextravasate from peripheral blood. These macrophages also phagocytose MTB, but they arealso unactivated and hence cant destroy the bacteria.Stage 3At this stage lymphocytes begin to infiltrate. The lymphocytes, specifically T-cells, recognizeprocessed and presented MTB antigen in context of MHC molecules. This results in T-cellactivation and the liberation of cytokines including gamma interferon (IFN). The liberationof IFN causes in the activation of macrophages. These activated macrophages are nowcapable of destroying MTB.It is at this stage that the individual becomes tuberculin-positive. This positive tuberculinreaction 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 antibodymediated immune (AMI) will not aid in the control of a MTB infection because MTB isintracellular and if extracellular, it is resistant to complement killing due to the high lipidconcentration in its cell wall.Although a CMI response is necessary to control an MTB infection, it is also responsible formuch of the pathology associated with tuberculosis. Activated macrophages may releaselytic enzymes and reactive intermediates that facilitate the development of immunepathology. Activated macrophages and T-cells also secrete cytokines that may also play arole in the development of immune pathology, including Interleukin 1 ( IL-l), tumor necrosisfactor (TNF), and gamma IFN.It is also at this stage that tubercle formation begins. The center of the tubercle ischaracterized 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 anoxicenvironment. MTB can, however, persist within these tubercles for extended periods.Stage 4Although many activated macrophages can be found surrounding the tubercles, many othermacrophages present remain unactivated or poorly activated. MTB uses these macrophagesto replicate, and hence, the tubercle grows.The growing tubercle may invade a bronchus. If this happens, MTB infection can spread toother parts of the lung. Similarly the tubercle may invade an artery or other blood supplyline. The hematogenous spread of MTB may result in extrapulmonary tuberculosisotherwise known as milliary tuberculosis. The name "milliary" is derived from the fact thatmetastasizing tubercles are about the same size as a millet seed, a grain commonly grown inAfrica. 18
  19. 19. 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 PMNs around MTB. Here the bacteriareplicate with virtually no resistance. This situation gives rise to the formation of a "softtubercle".2. Productive or granulomatous lesions occur when the host becomes hypersensitive totuberculoproteins. This situation gives rise to the formation of a "hard tubercle".Stage 5For unknown reasons, the caseous centers of the tubercles liquefy. This liquid is veryconducive to MTB growth, and the organism begins to rapidly multiply extracellularly. Aftertime, the large antigen load causes the walls of nearby bronchi to become necrotic andrupture. This results in cavity formation. This also allows MTB to spill into other airways andrapidly spread to other parts of the lung.As stated previously, only a very small percent of MTB infections result in disease, and evena smaller percentage of MTB infections progress to an advanced stage. Usually the host willbegin to control the infection at some point. When the primary lesion heals, it becomesfibrous 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, theGhon complex is readily visible upon chest X-ray.Small metastatic foci containing low numbers of MTB may also calcify. However, in manycases these foci will contain viable organisms. These foci are referred to Simon foci. TheSimon 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 isnt strong enough tokeep the infectious bacteria in check. Presence of the Mycobacterium tuberculosis bacteriacauses an immune response in which many types of white blood cells are recruited to siteswhere 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 conditionsleading to a weakened immune response can allow the bacteria to break out of the lesionand reactivate to develop into active TB. 19
  20. 20. Chapter 2M.Tuberculosis 20
  21. 21. 2.1 Morphology & Characteristics Mycobacterium tuberculosis "Captain among these Men of Death" (John Bunyon 1660) White Death White Plague Consumption Tuberculosis... Mycobacterium tuberculosis is a fairly large nonmotile rod-shaped bacterium distantlyrelated to the Actinomycetes. Many non pathogenic mycobacteria are components of thenormal flora of humans, found most often in dry and oily locales. The rods are 2-4micrometers in length and 0.2-0.5 um in width.Mycobacterium tuberculosis is an obligate aerobe. For this reason, in the classic case oftuberculosis, 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 slowgeneration time, 15-20 hours, a physiological characteristic that may contribute to itsvirulence.Two media are used to grow MTB Middlebrooks medium which is an agar based mediumand Lowenstein-Jensen medium which is an egg based medium. MTB colonies are smalland buff colored when grown on either medium. Both types of media contain inhibitors tokeep contaminants from out-growing MT. It takes 4-6 weeks to get visual colonies on eithertype of media.Chains of cells in smears made from in vitro-grown colonies often form distinctiveserpentine cords. This observation was first made by Robert Koch who associated cordfactor with virulent strains of the bacterium.Figure (2-1) Serpentine cord. Figure (2-2) M.Tuberculosis with ZN stain. Figure (2-3) M. tuberculosis Colonies. 21
  22. 22. MTB is not classified as either Gram-positive or Gram-negative because it does not havethe 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 asacid-fast bacteria due to their impermeability by certain dyes and stains. Despite this, oncestained, acid-fast bacteria will retain dyes when heated and treated with acidified organiccompounds. 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 withmethylene-blue or certain other dyes. Acid-fast bacilli appear pink in a contrastingbackground.In order to detect Mycobacterium tuberculosis in a sputum sample, an excess of 10,000organisms per ml of sputum are needed to visualize the bacilli with a 100X microscopeobjective (1000X mag). One acid-fast bacillus/slide is regarded as "suspicious" of an MTBinfection. 2.2 Cell Wall Structure The cell wall structure of Mycobacterium tuberculosis deserves special attentionbecause it is unique among procaryotes, and it is a major determinant of virulence for thebacterium. The cell wall complex contains peptidoglycan, but otherwise it is composed ofcomplex lipids. Over 60% of the mycobacterial cell wall is lipid. The lipid fraction of MTBscell 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 andCorynebacterium. 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 organismand affect permeability properties at the cell surface. Mycolic Acids are thought to be asignificant determinant of virulence in MTB. Probably, they prevent attack of themycobacteria 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 toxicto mammalian cells and is also an inhibitor of PMN migration. Cord factor is mostabundantly produced in virulent strains of MTB.Wax-D in the cell envelope is the major component of Freunds complete adjuvant (CFA). 22
  23. 23. 2.3 Mycobacterium Culture Culture of mycobacterium is the definitive method to detect bacilli. It is also moresensitive than examination of the smear. Approximately 10 acid-fast bacilli (AFB) permillimeter of a digested concentrated specimen are sufficient to detect the organisms byculture.Another advantage of culture is that it allows specific species identification and testing forrecognition 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 conventionalculture media.2.3.1 Conventional Growth Techniques Conventional solid media include the Löwenstein-Jensen medium, which is an egg-basedmedium, 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 requireincubation in 5-10% carbon dioxide for 3-8 weeks. These media usually have antibacterialantibiotics, which are slightly inhibitory for tubercle bacilli.2.3.2 Rapid Growth Techniques Because mycobacteria require 6-8 weeks for isolation fromconventional media, automated radiometric culture methods(eg, BACTEC) are increasingly used for the rapid growth ofmycobacteria. The methodology uses a liquid Middlebrook7H12 medium that contains radiometric palmitic acid labeledwith radioactive carbon-14 (14 C). Several antimicrobial agentsare added to this medium to prevent the growth ofnonmycobacterial contaminants. Production of14 CO2 by themetabolizing organisms provides a growth index for themycobacteria. Growth is generally detected within 9-16 days. Figure (2-4)SEPTI-CHEK AFB Culture BottleAnother rapid method for isolation of mycobacteria is SEPTICHEK. This nonradiometricapproach has a biphasic broth-based system that decreases the mean recovery time versusconventional 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 indicatemicrobial 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 culturecharacteristics, growth parameters, and other empiric tests. M tuberculosis produces heat-sensitive catalase, reduces nitrates, produces niacin, and grows slowly. Serpentine cordingis demonstrated on smears prepared from the BACTEC system. 23
  24. 24. Addition of p -nitro-acetyl-amino-hydroxy-propiophenone (NAP) inhibits the growth of Mtuberculosis complex (including M bovis and M africanum) but does not inhibit growth ofother 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-performanceliquid chromatography (HPLC). The unique mycolic acid pattern associated with the speciescan be detected by the chromatographic separation of the ester.A significant drawback of these chromatographic methods is the requirement of bacterialcolonies 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 moresensitive; thus, BACTEC broth culture can be used instead of conventional solid media. Thismay make the method comparable to the NAP and AccuProbe tests (see Nucleic AcidProbes). The expense of the initial equipment limits the availability of HPLC.2.3.3. ANucleic Acid Probes Because biochemical methods are time-consuming and laborious, nucleic acidhybridization using molecular probes has become widely accepted. The basic principle is theuse of a chemiluminescent, ester-labeled, single-strand DNA probe. A luminometer is usedto assess the chemiluminescence.Commercially available probes, including the AccuProbe technology, help advanceidentification 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 doesnot reliably differentiate between M tuberculosis and other members of the complex (eg, Mbovis). In addition, final identification to species level is required, because pyrazinamideshould 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. BNucleic Acid Amplification Tests Nucleic acid amplification techniques (eg, polymerase chain reaction [PCR]) allows thedirect 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 Mtuberculosis direct test and the AMPLICOR M tuberculosis test. The amplified M tuberculosisdirect test is an isothermal transcription-mediated amplification that targets RNA. TheAMPLICLOR test targets the DNA. The most commonly used target sequence for thedetection of M tuberculosis has been the insertion sequence IS6110. 24
  25. 25. Although amplification techniques are promising tools for the rapid diagnosis oftuberculosis (TB), several caveats remain. Contamination of samples by products ofprevious 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-positivecases exceed 95%, the sensitivity of smear-negative cases varies from 40% to 70%. Thus,discordance between the acid-fast smear result and the nucleic acid amplificationtechniques requires careful clinical appraisal and judgment. 2.4 Other Mycobacteria Species Reservoir Common clinical manifestation,comment Species always considered pathogens Mycobacteria Humans Pulmonary & disseminated tuberculosis tuberculosis Mycobacteria lepra Humans Leprosy Mycobacteria bovis Humans , cattle Tuberculosis-like disease Species potentially pathogens to humans (moderately ) Mycobacteria avium Soil , water , birds , fowl Disseminated , Pulmonary; common in complex , cattle , swine , AIDS patients environmentMycobacteria kansasii Table Water , cattle (2-1) other mycobacteria species Pulmonary , other sites Species potentially pathogens to humans (very rare )Mycobacteria africaum Humans , monkeys Pulmonary cultures ; resembles Mycobacteria tuberculosis Mycobacteria Humans , pet birds Blood in AIDS patients genavense Rapid growersMycobacteria abscessus Soil , water , animals Pulmonary infections 25Mycobacteria chelonae Soil , water , marine Cutaneous lesions most common life , animals
  26. 26. Chapter 3Diagnostic Laboratory Test 26
  27. 27. 3.1 Specimen Collection For Analysis The initial step in detection and isolation of the mycobacterium is to obtain appropriatespecimens 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 thedisease.Decontamination of other microorganisms in the specimens obtained may be performed bythe addition of sodium hydroxide, usually in combination with N -acetyl-L -cysteine. Otherbody fluids (eg, CSF, pleural fluid, peritoneal fluid) can also be centrifuged; the sediment canbe stained and evaluated for presence of acid-fast bacilli (AFB). CSF smear results arepositive in fewer than 10% of patients in some series. Enhancement of the yield may bepossible by staining any clot that may have formed in standing CSF specimens, as well asusing the sediment of a centrifuged specimen. Increased yield may also be obtained fromcisternal 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 thoseyounger than 6 years, gastric aspirates are used.Nasopharyngeal secretions and saliva are not acceptable. In older children, bronchialsecretions may be obtained by the stimulation of cough by an aerosol solution of propyleneglycol 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 thescarcity of the organisms in children compared with adults. An early morning sample shouldbe obtained before the child has had a chance to eat or ambulate, because these activitiesdilute the bronchial secretions accumulated during the night.Initially, the stomach contents should be aspirated, and then a small amount of sterilewater is injected through the orogastric tube. This aspirate should also be added to thespecimen.Because gastric acidity is poorly tolerated by the tubercle bacilli, neutralization of thespecimen should be performed immediately with 10% sodium carbonate or 40% anhydroussodium phosphate. Even with careful attention to detail and meticulous technique, thetubercle bacilli can be detected in only 70% of infants and in 30-40% of children withdisease. 27
  28. 28. 3.1.3 Bronchial Secretions Bronchoalveolar lavage may be used in older children (6 y or older). Bronchial secretionsmay be obtained by the stimulation of cough by an aerosol solution of propylene glycol in10% sodium chloride. This technique may also be used to provide bronchial secretions fordetection 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 preliminaryconfirmation of the diagnosis. Conventional methods include the Ziehl-Neelsen stainingmethod. The Kinyoun stain is modified to make heating unnecessary. Fluorochrome stains,such as auramine and rhodamine, are variations of the traditional stains. The majoradvantage of these methods is that slides can be screened faster, because the acid-fastmaterial 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 theinfectiousness of the patient and in determining the discontinuation of respiratory isolation.However, for reliably producing a positive result, smears require approximately 10,000organisms/mL. Therefore, in early stages of the disease or in children in whom the bacilli inthe respiratory secretions are sparse, the results may be negative. A single organism on aslide is highly suggestive and warrants further investigation.A significant drawback of AFB smears is that they cannot be used to differentiate Mtuberculosis from other acid-fast organisms such as other mycobacterial organisms orNocardia species. Figure (3-2) A sputum smear stained with auramine. Figure (3-1) AFB smear (Notethe small, purple, rod-shaped bacilli). 28
  29. 29. 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 procedure3.2.2 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.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... 29
  30. 30. 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 combinations K530 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 nanometers3.2.3 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: 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. 30
  31. 31. 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 patients general health and find otherfactors 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 StudiesFigure (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 Mycobacteriumtuberculosis organisms from a specimen taken from the patient (most often sputum, butmay also include pus, CSF, biopsied tissue, etc.). A diagnosis made other than by culturemay only be classified as "probable" or "presumed". For a diagnosis negating the possibilityof tuberculosis infection, most protocols require that two separate cultures both testnegative.3.4.1 Sputum Smear & Culture Sputum smears and cultures should be done for acid-fast bacilli if the patient isproducing sputum. The preferred method for this is fluorescence microscopy (auramine-rhodamine staining), which is more sensitive than conventional Ziehl-Neelsen staining. In 31
  32. 32. cases where there is no spontaneous sputum production, a sample can be induced, usuallyby nebulized inhalation of a saline or saline with bronchodilator solution. A comparativestudy found that inducing three sputum samples is more sensitive than three gastricwashings.3.4.2 Alternative Sampling In patients incapable of producing a sputum sample, common alternative sample sourcesfor diagnosing pulmonary tuberculosis include gastric washings, laryngeal swab,bronchoscopy (with bronchoalveolar lavage, bronchial washings, and/or transbronchialbiopsy), and fine needle aspiration (transtracheal or transbronchial). In some cases, a moreinvasive technique is necessary, including tissue biopsy during mediastinoscopy orthoracoscopy.3.4.3 PCR Other mycobacteria are also acid-fast. If the smear is positive, PCR or gene probe testscan 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 AFBcan distinguish the various forms of mycobacteria, although results from this may take fourto eight weeks for a conclusive answer. New automated systems that are faster include theMB/BacT, BACTEC 9000, and the Mycobacterial Growth Indicator Tube (MGIT). TheMicroscopic Observation Drug Susceptibility assay culture may be a faster and moreaccurate method.3.5 Radiography3.5.1Chest X-ray Tuberculosis creates cavities visible in x-rays like this one in thepatients right upper lobe figure (3-6) chest x-rays. In active pulmonary TB, infiltrates or consolidations and/or cavitiesare often seen in the upper lungs with or without mediastinal or hilarlymphadenopathy or pleural effusions ( tuberculous pleurisy). However,lesions may appear anywhere in the lungs. In disseminated TB a patternof many tiny nodules throughout the lung fields is common - the socalled miliary TB. In HIV and other immunosuppressed persons, any abnormality mayindicate TB or the chest X-ray may even appear entirely normal.Figure (3-6) chest x-rays 32
  33. 33. 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 aperson 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 discernibleby a chest x-ray.3.5.2 Abreugraphy A variant of the chest X-Ray, abreugraphy (from the name of its inventor, Dr. ManuelDias de Abreu) was a small radiographic image, also called miniature mass radiography(MMR) or miniature chest radiograph. Though its resolution is limited (it doesnt allow thediagnosis 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 extensivelyutilized in some countries, in the 1950s. For example, in Brazil and in Japan, tuberculosisprevention laws went into effect, obligating ca. 60% of the population to undergo MMRscreening.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 immigrationapplicants.3.6 Immunological Test3.6.1 ALS Assay Antibodies from Lymphocyte Secretion or Antibody in Lymphocyte Supernatant or (ALSAssay) is an immunological assay to detect active diseases like tuberculosis, cholera, typhoidetc. Recently, ALS assay nods the scientific community as it is rapidly used for diagnosis ofTuberculosis. The principal is based on the secretion of antibody from in vivo activatedplasma 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)Two tests are available: the Mantoux & Heaf tests.3.6.2. A Mantoux Skin Test 33
  34. 34. 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 AmericanThoracic 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 pointscale. 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 (dependingupon the persons risk factors) to 10 Mantoux units is considered a positive result, indicatingTB 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• 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 34
  35. 35.  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-yearperiod, 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 beenimmunized with BCG. The US recommendation is that in administering and interpreting theMantoux test, previous BCG vaccination should be ignored; the UK recommendation is thatinterferon-γ tests should be used to help interpret positive tuberculin tests, also, the UK donot recommend serial tuberculin skin testing in people who have had BCG (a key part of theUS strategy). In their guidelines on the use of QuantiFERON Gold the US Centers for DiseaseControl and Prevention state that whereas Quantiferon Gold is not affected by BCGinoculation tuberculin tests can be affected. In general the US approach is likely to result inmore false positives and more unnecessary treatment with potentially toxic drugs; the UKapproach is as sensitive in theory and should also be more specific, because of the use ofinterferon-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, ifany 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.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 acidsequence they detect and vary in their accuracy. The two most common commerciallyavailable 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 HealthTechnology Assessment Programme concluded that "NAAT test accuracy to be far superiorwhen applied to respiratory samples as opposed to other specimens. Although the results 35
  36. 36. were not statistically significant, the AMTD test appears to perform better than othercurrently available commercial tests.In a more recent before-after observational study, found that use of the MTD test reduceinappropriate tuberculosis therapy. The study found the accuracy of the MTD test asfollows:Overall• sensitivity 92%• specificity 99%Smear positive patients• sensitivity 99%• specificity 98%Smear negative patients• sensitivity 62%• specificity 99%3.8 Interferon-γ Release Assays Interferon-γ (interferon-gamma) release assays (IGRAs) are exciting new developmentsin TB infection testing. IGRAs are based on the ability of the Mycobacterium tuberculosisantigens 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 presentin non-tuberculous mycobacteria or in any BCG vaccine variant, these tests can distinguishlatent tuberculosis infection (LTBI).The blood tests QuantiFERON-TB Gold In Tube and T-SPOT.TB use these antigens to detectpeople with tuberculosis. Lymphocytes from the patients blood are incubated with theantigens. These tests are called interferon γ tests and are not equivalent. If the patient hasbeen exposed to tuberculosis before, T lymphocytes produce interferon γ in response. TheQuantiFERON-TB Gold In Tube uses an ELISA format to detect the whole blood production ofinterferon γ with great sensitivity (89%).The distinction between the tests is thatQuantiFERON-TB Gold quantifies the total amount of interferon γ when whole blood isexposed to the antigens(ESAT-6,CFP-10 and TB 7.7(p4)), whereas Guidelines for the use ofthe FDA approved QuantiFERON-TB Gold were released by the CDC in December 2005. InOctober 2007, the FDA gave approval of QuantiFERON-TB Gold In Tube for use in the UnitedStates.The enzyme-linked immunospot assay (ELISPOT) is another blood test available in the UKthat may replace the skin test for diagnosis. T-SPOT.TB, a type of ELISPOT assay, counts thenumber of activated T lymphocytes that secrete interferon γ. 36
  37. 37. For diagnosing latent TB, three systematic reviews of IGRAs concluded the tests notedexcellent 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-TBGold 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 fromboth developed and developing countries, QuantiFERON-TB Gold In Tube had a pooledsensitivity for active TB of 81% and specificity of 99.2%, whereas T-SPOT.TB had a pooledsensitivity of 87.5% and specificity of 86.3%. In head-to-head comparisons, the sensitivity ofIGRAs surpassed TST. The authors concluded that IGRAs are "superior to the TST fordetecting confirmed active TB disease. Table (3-1) characteristics of methods for clinical mycobacteriology 37
  38. 38. Chapter 4Treatment & Prevention 38
  39. 39. 4.1 Treatment A person with a positive skin test, a normal chest X-ray, and no symptoms most likelyhas only a few TB germs in an inactive state and is not contagious. Nevertheless, treatmentwith an antibiotic may be recommended for this person to prevent the TB from turning intoan active infection. The antibiotic used for this purpose is called isoniazid (INH). If taken forsix to 12 months, it will prevent the TB from becoming active in the future. In fact, if aperson with a positive skin test does not take INH, there is a 5%-10% lifelong risk that the TBwill become active.Taking isoniazid can be inadvisable (contraindicated) during pregnancy or for those sufferingfrom alcoholism or liver disease. Also, isoniazid can have side effects. The side effects occurinfrequently, but a rash can develop, and the individual can feel tired or irritable. Liverdamage 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 evenbe fatal. It is important therefore, for the doctor to monitor a patients liver by periodicallyordering blood tests called "liver function tests" during the course of INH therapy. Anotherside effect of INH is a decreased sensation in the extremities referred to as a peripheralneuropathy. This can be avoided by taking vitamin B6 (pyridoxine), and this is oftenprescribed along with INH.A person with a positive skin test along with an abnormal chest X-ray and sputumevidencing TB bacteria has active TB and is contagious. As already mentioned, active TBusually 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 totreat active TB in conjunction with isoniazid (INH). Four drugs are often taken for the firsttwo months of therapy to help kill any potentially resistant strains of bacteria. Then thenumber 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 drugthat is given by injection, may be used as well, particularly when the disease is extensiveand/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 ofTB is dependent largely on the compliance of the patient. Indeed, the failure of a patient totake the medications as prescribed is the most important cause of failure to cure the TB 39
  40. 40. infection. In some locations, the health department demands direct monitoring of patientcompliance with therapy.Surgery on the lungs may be indicated to help cure TB when medication has failed, but inthis day and age, surgery for TB is unusual. Treatment with appropriate antibiotics willusually cure the TB. Without treatment, however, tuberculosis can be a lethal infection.Therefore, early diagnosis is important. Those individuals who have been exposed to aperson with TB, or suspect that they have been, should be examined by a doctor for signs ofTB and screened with a TB skin test. 4.2 Multi Drug Resistance M. Tuberculosis Drug-resistant TB (TB that does not respond to drug treatment) has become a veryserious problem in recent years in certain populations. For example, INH-resistant TB is seenamong patients from Southeast Asia. The presence of INH-like substances in the coughsyrups in that part of the world may play a role in causing the INH resistance. Drug-resistantcases are also often seen in prison populations. However, the major reason for thedevelopment of resistance is poorly managed TB care. This can result from poor patientcompliance, inappropriate dosing or prescribing of medication, poorly formulatedmedications, 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-resistanttuberculosis (XDR-TB) has emerged. These bacteria are also resistant to three or more ofthe second-line treatment drugs.Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB. XDR TB is definedas TB which is resistant to isoniazid and rifampin, plus resistant to any fluoroquinolone andat least one of three injectable second-line drugs (i.e., amikacin, kanamycin, orcapreomycin).Because XDR TB is resistant to first-line and secondline drugs, patients are left withtreatment options that are much less effective.XDR-TB is seen throughout the world but is most frequently seen in the countries of theformer 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 thedevelopment of proper laboratories for detection of resistant cases. When drug-resistantcases are found, prompt, appropriate treatment is required. This will prevent furthertransmission. Collaboration of HIV and TB care will also help limit the spread of tuberculosis,both sensitive and resistant strains. 40
  41. 41. 4.3 Epidemiology & Statistics People with silicosis have an approximately 30-fold greater risk for developing TB. Silicaparticles irritate the respiratory system, causing immunogenic responses such asphagocytosis, which results in high lymphatic vessel deposits. It is probably this interferenceand 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 timesgreater than persons without diabetes mellitus, and this risk is likely to be greater inpersons with insulin-dependent or poorly controlled diabetes.Other clinical conditions that have been associated with active TB include gastrectomy withattendant weight loss and malabsorption, jejunoileal bypass, renal and cardiactransplantation, 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 effectof various indoor or outdoor air pollutants on the disease would be necessary. Somepossible indoor sources of silica include paint, concrete, and Portland cement. Crystallinesilica is found in concrete, masonry, sandstone, rock, paint, and other abrasives. Thecutting, breaking, crushing, drilling, grinding, or abrasive blasting of these materials mayproduce 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 theyhave a poorer response to treatment, possibly due to poorer drug absorption.Diabetes increases the risk of TB three-fold. The correlation between diabetes mellitus andTB is concerning for public health because it shows a distinct connection between acontagious 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 contactwith the bacteria. Diabetics do not have an increased risk of contracting latent tuberculosis 41
  42. 42. but studies have shown that people with diabetes mellitus are more likely to move from alatent 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 ofprevious TB, showing fibrotic lesions and nodules, prolonged corticosteroid therapy andother immunosuppressive therapy, compromised immune system (30–40% of people withAIDS worldwide also have TB), hematologic and reticuloendothelial diseases, such asleukemia and Hodgkins disease, end-stage kidney disease, intestinal bypass, chronicmalabsorption 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 oftwins 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. Specificgene polymorphisms in IL12B have been linked to tuberculosis susceptibility.Some drugs, including rheumatoid arthritis drugs that work by blocking tumor necrosisfactor-alpha (an inflammation-causing cytokine), raise the risk of activating a latentinfection due to the importance of this cytokine in the immune defense against TB. 42
  43. 43. 4.4 TB control in JordanThe WHO collaborative programmeTUBERCULOSIS CONTROLSituation analysisJordan has a low incidence of tuberculosis. The estimated incidence rate of all TB casesis 7 per 100,000 populations. Every year, 400 people are estimated to develop TB in thecountry. 30 % of the cases occur among the productive age groups of the community (agedbetween 15 to 54 years old).TB control in Jordan has made considerable progress during the last few years. The NationalTB Programme (NTP) started implementing DOTS in 1998, and achieved the RegionalTargets of DOTS ALL OVER in 1998. In 2000, 303 cases of TB were notified in DOTS areas, ofwhich 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 TBAssociation is providing support to the national TB programme through financial aid to TBpatients, health education and in numerous other activities. Ministry of Defence has startedusing DOTS in their health services. Jordan also made good progress in the TB EliminationInitiative by lowering the incidence rate of smear positive new cases to 7 per 100,000populations.Main achievements 43
  44. 44. 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 & ControlControl 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. 44
  45. 45. Precotions Polices  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) strategywas launched formally as Revised National TB Control programme in India in 1997 afterpilot testing from 1993-1996. Since then DOTS has been widely advocated and successfullyapplied. 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 andcomplete treatment of the patient lies with the health system. In 2006, the new stop TB strategy was recommended internationally by WHO. Thecomponents of the new stop TB strategy are the following: 45
  46. 46. 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 researchReferences 1. G.Brooks, K.Carrroll, J.Butel, S.Melnicks (Medical Microbiology 24th Edition) Jawetz, Melnicks Dahlbergs (2010). 2. Landau, Elaine. Tuberculosis. New York: F. Watts, 1995 http://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 Goals WHO. Geneva, World Health Organization, 2006b (WHO/HTM/STB/2006.37). 5. Global burden of tuberculosis: estimated incidence, prevalence and mortality by country Dye C et al. Global burden of tuberculosis: estimated incidence, prevalence and mortality by country. Journal of the American Medical Association, 1999. 46
  47. 47. 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 May9. 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 47