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pulmonary Tuberculosis
1. 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
1
2. November 2011
Al-Balqa' Applied University
Zarka University College
Faculty of Medical Analysis
Allied Medical Science Department
Pulmonary Tuberculosis
Supervisor:
Dr. Waleed Al-
Momani.
2
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. Abstract
T uberculosis 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.
4
6. 3.5 Radiography 32
3.5.1 Chest X-Ray 32
3.5.2 Abreugraphy 32
3.6 Immunological Test
3.6.1 ALS Assay 33
3.6.2 Tuberculin Skin Test (TST) 33
3.6.2. A Mantoux Skin Test 33
3.6.2.B Heaf Test 34
3.6.2.C BCG Vaccine And Tuberculin Skin Test 35
(Nucleic Acid Amplification Tests (Naat 3.7 35
γ -Interferon Release Assays 3.8 36
Chapter 4 Treatment & Prevention
Treatment 4.1 39
Multi Drug Resistance Tuberculosis 4.2 40
Epidemiology & Statistics 4.3 41
Prevention & Control 4.4 43
TB 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. List of Tables
TABLE (1-1) : M.Tuberculosis Species Complex 11
TABLE (1-2) : Tuberculosis Infection Vs. Disease 16
TABLE (2-1) : Other Mycobacteria Species 25
TABLE (3-1) : Characteristics Of Methods For Clinical Mycobacteriology 37
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.
7
9. 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.
9
10. 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.
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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11. 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.
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. Table (1-1) Mycobacterium tuberculosis complex
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
12
13. "osseous tuberculosis", a form of Osteomyelitis (as a complication of tuberculosis). An
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)
13
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 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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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 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
15
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 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+).
16
17. 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.
17
18. 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.
18
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 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.
19
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 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.
Figure (2-1) Serpentine cord. Figure (2-2) M.Tuberculosis with ZN stain. Figure (2-3) M. tuberculosis Colonies.
21
22. 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
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).
22
23. 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.
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.
23
24. 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.
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.
24
25. 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,
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 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
environment
Mycobacteria 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 growers
Mycobacteria abscessus Soil , water , animals Pulmonary infections
25
Mycobacteria chelonae Soil , water , marine Cutaneous lesions most common
life , animals
27. 3.1 Specimen Collection For Analysis
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.
27
28. 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
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 (3-2) A sputum smear stained
with auramine.
Figure (3-1) AFB smear (Note
the small, purple, rod-shaped bacilli).
28
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 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.
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. 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 filter
Mercury 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:
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. 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
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
31
32. 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
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
32
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 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)
Two tests are available: the Mantoux & Heaf tests.
3.6.2. A Mantoux Skin Test
33
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 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
• 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. 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.
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
35
36. 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-γ Release Assays
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
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.
36
37. 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.
Table (3-1) characteristics of methods for clinical mycobacteriology
37
39. 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
39
40. 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.
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.
40
41. 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
41
42. 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
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.
42
43. 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
43
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 & 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.
44
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) 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:
45
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 research
References
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The End
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