Professional Documents
Culture Documents
Tuberculosis
(Mycobacterium Tuberculosis)
Written By:
Abdullah Said Al-Barakat
U.N.:
424101197
Supervision:
Dr. Jamal Eid Al-Said
Introduction 5
Mycobacterium Tuberculosis
Morphology of Mycobacterium Tuberculosis 8
Virulence Factors of Mycobacterium Tuberculosis 11
1- Cord Factor(s) 11
2- Mycobacterial Sulfolipids 11
3- Mycoside 12
Transmission Of Mycobacterium Tuberculosis 13
Pathogenesis
Pathogenesis of Tuberculosis 15
1- Primary Tuberculosis 15
2- Post-primary Tuberculosis 18
3- Immunocompromised individuals 20
Symptoms Of Tuberculosis 21
Immunologic response
Immunologic Response Against Tuberculosis 23
Diagnosis Of Tuberculosis
Diagnosis Of Tuberculosis 30
1- Sputum Examination 30
1-1- Preparation, Staining and Microscopic 30
Examination
2- Culture Method 37
2-1- Preparation of Specimen 37
2-2- Incubation Conditions 37
2-3- Automated System 38
2-4- Identification 38
3- X-ray 39
4- Tuberculin Test 40
4-1- Description 40
4-2- Preparation 40
4-3- Aftercare 40
4-4- Risks 41
4-5- Normal results 41
4-6- Abnormal Results 42
5- Polymerase Chain Reaction 43
New Diagnostic Method 46
1- Intended Used 46
2- Summary and Explanation of The Test 46
3- Principle of The Assay 47
4- Reagent and Storage 48
4-1- Peptide and Control Antigens 48
4-2- ELISA Component 48
4-3- Storage Instructions 48
5- Specimen Collection and Handling 49
6- Directions of Use 49
6-1- Stage One 49
6-1- Stage Two 51
7- Interpretation of Results 55
8- Warnings and Precaution 56
8-1- Warnings 56
8-3- Precaution 56
BCG Vaccine
BCG Vaccine of Tuberculosis 59
1- BCG Vaccine 59
2- Storing and Validity 59
3- Indication 59
4- Complication 60
5- BCG Vaccine and Other Vaccines 60
6- Administration and Dosage 60
7- Site 61
Treatment
Treatment of Tuberculosis 63
1- General Roles of Tuberculosis Treatment 63
2- Directly Observed Treatment, Short Course (DOTS) 63
3- Phases of Treatment 63
4- Hospitalization 64
5- Duration of Treatment 64
6- General Procedure that should be followed during 64
Treatment
7- Categories of Treatment 65
8-1- CAT 1 65
8-2- CAT 2 65
8-3- CAT 3 66
8-4- CAT 4 66
67
Extrapulmonary Tuberculosis
1- Hepatic 69
1-1- Clinical 69
1-2- Diagnosis 70
2- Meninges 71
2-1- Clinical 71
2-2- Diagnosis 72
Conclusion 72
References 76
Figures References 83
Introduction
Tuberculosis (TB) is an infection caused by a germ called the tubercle
bacillus or Mycobacterium tuberculosis. Until effective anti-tuberculosis
drugs were introduced about 50 years ago, TB was one of the main causes
of death.
Tuberculosis most commonly attacks the lungs (as pulmonary TB) but
can also affect the central nervous system, the lymphatic system, the
circulatory system, the genitourinary system, bones, joints and even the
skin. Other mycobacteria such as Mycobacterium bovis, Mycobacterium
africanum, Mycobacterium canetti, and Mycobacterium microti can also
cause tuberculosis, but these species do not usually infect healthy adults.
The mycobacteria are rode – shaped that do not form spores. Although
they do not stain readily, once stained they resist decolorization by acid
or alcohol and are therefore called "acid-fast" bacilli.
In tissue, tubercle bacilli are thin straight rods measuring 0.4×3 um.
On artificial media, coccid and filamentous forms are seen with variable
morphology from one species to another. Mycobacteria can not be
classified as either gram-positive or gram-negative. Once stained by basic
dyes they can not be decolorized by alcohol, regardless of treatment with
iodine.
The mycolic acids containing extremely long (C78-C90) side chains are
joined to the muramic acid moiety of the peptidoglycan by
phosphodiester bridges and to arabinogalactan by esterified glycolipid
linkages. Species variations are characterized by variation in sugar
substitution in the glycolipids or peptidoglycolipids. The mycobacterial
cell wall is acid fast. This important property allows differential staining
in contaminated clinical specimens such as sputum.
1- Cord factor(s)
The problem with ascribing cord factor with a major role in virulence
is its occurrence in nonpathogenic as well as pathogenic species of
mycobacteria. (6)
SLs kill mice when injected intrapertioneally and enhance the toxicity
of cord factor. (8)
The SLs inhibit the fusion of MTB phagosome and lysosome, thus
allowing MTB to evade host microbcidal molecules. (10)
The SLs although inhibition of phagosome-lysosome fusion also may
be mediated by other molecules, such as ammonia produced by MTB. 7
3- Mycoside:
Other factor
Sigma factors, which are small transcription factors, regulate the
transcriptional activity of MTB during its adaptive states, and may be
indispensable for its virulence. (14)
Transmission of Mycobacterium tuberculosis
When people suffering from active pulmonary TB coughs, sneeze,
speak, kiss, or spit, they expel infectious aerosol droplets 0.5 to 5 um in
diameter. A single sneeze, for instance, can release up to 40,000 droplets.
(15)
The Transmission can only occur from people with active-not latent-TB.
The probability of transmission from one to another depends upon the
number of infectious droplets expelled by a carrier, the effectiveness of
ventilation, the duration of exposure, and the virulence of the
M.tuberculosis strain. (18)
Pathogenesis of Tuberculosis
The tubercle bacillus owes its virulence to its ability to survive within
the macrophage rather than to the production of a toxic substance. The
mechanism of virulence is poorly understood and is almost certainly
multifactorial. The immune response to the bacillus is of the cell-
mediated type, which, if mediated by Th1 T helper cells, leads to
protective immunity, but the presence of Th2 cells facilitates tissue-
destroying hypersensitivity reactions and progression of the disease
process. The nature of the immune responses following infection change
with time so that human tuberculosis is divisible into primary and post-
primary forms with quite different pathological features.
1- Primary Tuberculosis
The site of the initial infection is usually the lung. Following the
inhalation of bacilli. These bacilli are engulfed by alveolar macrophage in
which they replicate to form the initial lesion or Ghon focus. Some bacilli
are carried in phagocytic cells to the hilar lymph nodes where additional
foci of infection develop. The Ghon focus, together with the enlarged
hilar lymph nodes, form the primary complex. In addition, bacilli are
seeded by further lymphatic and haematogenous dissemination in many
organs and tissue, including other parts of the lung. When the
bacilli enter the mouth, as when drinking milk containing M. bovis, the
primary complexes involve the tonsil and cervical nodes or the intestine,
often the ileocaecal region, and the mesenteric lymph nodes. Likewise,
the primary focus may be in skin, with involvement of the regional lymph
nodes. This form of tuberculosis was an occupational disease of
anatomists and pathologists and was termed prosector's wart.
Within about 10 days of infection, clones of antigen-specific T
lymphocyte are produced. These release cytokines, notably interferon- ,
which activate macrophage and cause them to form a compact cluster,
and cause them to form a compact cluster, or granuloma, around the foci
of infection. These activated macrophages are termed epithelioid cells.
Some of them fuse to form multinucleate giant cells. The center of the
granuloma contains a mixture of necrotic tissue and dead macrophages,
which, form its cheese-like appearance and consistency, is referred to as
caseation.
The interior of the tuberculoma is acidic and anoxic and contains few
viable tubercle bacilli. Eventually, however, the expending lesion erodes
through the wall of a bronchus, the liquefied contents are discharged and
a well-aerated activity is formed. The atmosphere of the lung, with a high
carbon dioxide level, is ideal for supporting the growth of the bacilli, and
huge numbers of these are found in the cavity walls. For this reason,
closure of the cavities by collapsing the lung, either by artificial
pneumothorax or by excising large portions of the chest wall, was a
standard treatment for tuberculosis in the pre-chemotherapy area.
Figure 7: Infected lung show the cavities
Once the cavity is formed, large numbers of bacilli gain access to the
sputum, and the patient becomes an open or infectious case. This is a
good example of the transmissibility of and a pathogen being dependent
upon the host's immune response to infection. Surprisingly, about 20% of
cases of open cavitating tuberculosis resolve without treatment.
When the disease becomes active, 75% of the cases are pulmonary
TB. Symptoms include chest pain, coughing up blood, and a productive,
prolonged cough for more than three weeks. Systemic symptoms include
fever, chills, night sweats, appetite loss, weight loss, pallor, and often to
tendency to fatigue very easily.
In the other 25% of active cases, the infection moves from the lung,
causing other types of TB more common in immunosuppresed persons
and young children. Extrapulmonary infection sites include pleura, the
central nervous system in meningitis, the lymphatic system in scrofula of
the neck, the genitourinary system is urogenital tuberculosis, and bones
and joints in Pott's disease of the spine. An especially serious form is
disseminated TB, more commonly Known as miliary tuberculosis.
Although extrapulmonary TB is not contagious, it may co-exist with
pulmonary TB, which is contagious. (96)
Immunologic Response against Tuberculosis
Since Tb is basically a pulmonary disease, the lung is the point of
entry for the microorganism and the principle manifestation site of the
infection. Immediately after a primary infection, air particles, alveolar
macrophages, and dendritic cells, this phagocytosed the M. tuberculosis;
migrate through the lymphatic system toward the regional lymph node,
forming the Ghon complex. Simultaneously, phagocytic cell can
penetrate the pulmonary parenchyma, initiating an inflammatory focus to
which other macrophages will be attracted. In this case, microorganism
initiates the formation of a granuloma, coordinated by T lymphocytes.
The T cells granulomas, indispensable to the formation of stable
granulomas, contacting mononuclear phagocytes and influencing their
differentiation and activation status. The M. tuberculosis is contained in
the granuloma, and can persist in the lesions for decades, in latent form,
without triggering the disease.
Also they said the cytotoxic T cells (CD8+), which recognize from the
cytoplasm (tumor or viral), also participate in the immune response to M.
tuberculosis. (21)
However, it has been shown that mice depleted of NK1.1 cells do not
present greater susceptibility to mycobacterial infection. (27)
Constituting the principle immune response, Th1 cells are necessary for
the control of the chronic phase of the infection, due to the effect that IL-
2 and IFN- have on T cells and macrophages. Produced by dendritic
cells and macrophages, IL-12 is active in T cells, forming a link between
the innate and acquired responses. Individuals with mutations in genes
IL-12p40 and IL-12R present reduced T-cell production of IFN- and are
more susceptible to infections disseminated by the bacilli Calmette-
Guerin (BCG) vaccine and M. avium. (30)
These levels are even lower in patients with advanced pulmonary disease.
(34)
General rules:
* A trained person should supervise collection of the sample, as the
sample collected under supervision is better than that collected without.
* The sample must be collected either out side in the open air or in a good
ventilated room specified for this purpose.
e- Give the patient a new container and make quite sure that he has
understood that he must spit into the container as soon as he coughs up
sputum in morning.
Microscopy:
a- Sputum samples should be examined by direct microscopy using light
microscope and Ziehl Nelseen Stain. A trained laboratory specialist or a
technician should examine the sputum.
* Fix the sample by passing it three times through the flam. The
smear must be uppermost and not facing the flam (never overheat the
slide).
1-1-4 Staining:
b- Cover the whole surface of the slides with Ziehl's carbol fuchsin.
c- Heat very gently until vapor rises, use the flam of Bunsen-burner or of
a wad of cotton-wool in alcohol fixed on the end of a metal rod or a fairy
strong stick of wood.
In no case must the stain boil or dry on the slide. If the stain
accidentally runs away, add more and heat again. Leave the warm stain
for five minutes.
Decolorization:
a- With the forceps, remove the filter paper and deposit them in the waste
receptacle.
c- Replace all slides on the slide-rack and cover each one individually
with 25% sulphuric acid for three minutes.
e- Decolorize again for 1-3 minutes as in (c) above until all color has
practically disappeared.
Counter-staining:
a- Replace decolorized, rinsed slides on slide-rack and flood smear with
0.3% methylene blue counter-stain for 60 minutes.
Examination by microscopy:
For the examination of stained specimens, a binocular microscope is
most convenient, with an immersion objective (X100) and eye piece of
moderate magnification (X6 or X8). Nevertheless, if there is no
electricity, and in hot or humid conditions, a monocular microscope
might be better, because there are fewer surfaces to be attacked by fungi.
Put a drop of immersion oil on the left edge of the stained smear (near
the engraved number) and place the slide on the microscope stage. To
avoid possible contamination of the immersion oil, do not touch the slide
with oil applicator, but permit the drop of oil to fall freely onto the slide.
With the macrometric screw, lower the immersion lens, keeping
continuous watch until it touches the drop of oil. Looking through the eye
pieces bring the immersion lens slowly upwards, by means of the
micrometric screw. All during the reading, the correct focusing is ensured
by using the micrometric screw.
Technique of reading:
Examine at least 100 microscopic fields. For a skilled microscopist,
this will take 5 minutes.
Tubercle bacilli look like red rods, slightly curved, more or less
granular, isolated, in pairs or in groups, standing out clearly against the
blue background. Count the number of AFB and report this number in the
notebook.
Figure 9: Mycobacterium tuberculosis - Ziehl Nelseen Stain
At the end of examination, take the slide from the microscope stage,
check the identification number engraved on it, and enter the result of the
examination in the lat column of the dispatch list. Dip the slide into
toluene (or xylol) to remove the immersion oil and place it in box foe
examined slides.
Before examining the next slide, wipe the immersion lens with a piece
of clean cotton. (42)
c- Cover the fixed smear with the auramine-phenol stain for 10 minutes.
g- Cover the smear with the potassium permanganate solution for about
10 seconds, followed by several rinses with clean water.
d- Wipe the back of the slide clean and place it in a draining rack for the
smear to dry. Do not blot dry. To prevent fading of the fluorescence,
protect the stained smear from sunlight and bright light.
Result:
2- Culture method
As sputum and certain other specimens frequently contain many
bacteria and fungi that would rapidly overgrow any mycobacteria on the
culture media, these must be destroyed. Decontamination methods make
use of the relatively high resistance of mycobacteria to acids, alkalis and
certain disinfectants.
2-4 Identification:
The first step in identification is to determine whether an isolate is a
member of the M. tuberculosis complex. These organisms:
* Grow slowly.
4-1 Description
TB skin tests are usually given at a clinic, hospital, or doctor's office.
Some times the tests are given at schools or workplaces and may be a pre-
employment requirement. Many cities provide free TB skin tests and
follow up care. The Mantoux PPD tuberculin skin test involves injecting
a very small amount of a substance called PPD tuberculin just under the
top layer of the skin (intracutaneously). Tuberculin is a mixture of
antigens obtained from the culture of M. tuberculosis. Antigens are
foreign particles or proteins that stimulate the immune system to produce
antibodies. The latter of PPD it is mean Purified Protein Derivative. The
latter is the preferred testing substance. The test is usually given on the
inside of the forearm about halfway between the wrist and the elbow,
where a small bubble will form as the tuberculin is injected. The skin test
takes just a minute to administer.
4-2 Preparation
There is no special preparation needed before a TB skin test. A brief
personal history will be taken to determine whether the person has had
tuberculosis or a TB test before, has been in close contact with anyone
with TB, or has any significant risk factors. Directly before the test, the
skin on the arm at the injection site is usually cleaned with an alcohol
swab and allowed to air dry.
4-3 Aftercare
After having a TB skin test, it is extremely important to make sure that
the patient keeps the appointment to have the test reaction read. The
patient is instructed to keep the test site clean, uncovered, and to not
scratch or rub the area. Should severe swelling, itching, or pain occur, or
if the patient has trouble breathing, the clinic or health care provider
should be contacted immediately.
4-4 Risks
The risk of an adverse reaction is very low. Occasionally, an
individual who has been exposed to the TB bacteria will develop a large
reaction in which the arm swells and is uncomfortable. This reaction
should disappear in two weeks. A sore might develop where the injection
was given, or a fever could occur, but these are extremely rare reactions.
First, four aliquots of herpanised whole blood are incubated with either
ESAT-6, CFP- 10, Mitogen or Nil control antigens.
The Nil samples adjust for background, heterophile antibody effects, and
non-specific IFN- in blood samples. The IFN- level of the nil samples
is subtracted from the IFN- levels of the TB peptide antigens and
Mitogen control.
*Time Required for Performing Assay:
Procedure
1. Blood samples must be evenly mixed before aliquoting. Use a roller-
rocker or invert tubes 20 times immediately prior to dispensing.
3. Prior to use, mix each stimulation antigen well. Use undiluted. Holding
the dropper bottle vertically, carefully add 3 drops of each antigen to the
appropriate wells containing blood.
FIGURE 16: Recommended layout for dispensing Blood and
Stimulation Antigens into 24 Well Culture Plates
Procedure
1. All plasma samples and reagents, except for Conjugate 100X
Concentrate, must be brought to room temperature (22°C ± 5°C) before
use. Allow at least 60 minutes for equilibration.
2. Remove strips that are not required from the frame, reseal in the foil
pouch, and return to the refrigerator for storage until required.
Allow at least one strip for the QuantiFERON®-TB Gold Standards and
sufficient strips for the number of subjects being tested.
After use, retain frame and lid for use with remaining strips.
• Prepare fresh dilutions of the Kit Standard for each ELISA session.
FIGURE 17: Preparation of Standard Curve
9. Cover each plate with a lid and incubate at room temperature (22°C ±
5°C) for 120 ± 5 minutes.
• Plates should not be exposed to direct sunlight during incubation.
10. During the incubation, dilute one part Wash Buffer 20X Concentrate
with 19 parts deionised or distilled water and mix thoroughly. Sufficient
Wash Buffer 20X Concentrate has been provided to prepare 2L of
Working Strength wash buffer.
Wash wells with 400 L of Working Strength wash buffer for at least 6
cycles. An automated plate washer is recommended.
• Thorough washing is very important to the performance of the assay.
Ensure each well is completely filled with wash buffer to the top of the
well for each wash cycle. A soak period of at least 5 seconds between
each cycle is recommended.
• Standard laboratory disinfectant should be added to the effluent
reservoir, and established procedures followed for the decontamination of
potentially infectious material.
11. Tap plates face down on absorbent towel to remove residual wash
buffer. Add 100 L of Enzyme Substrate Solution to each well and mix
thoroughly using a microplate shaker.
12. Cover each plate with a lid and incubate at room temperature (22°C ±
5°C) for 30 minutes.
• Plates should not be exposed to direct sunlight during incubation.
14. Measure the Optical Density (OD) of each well within 5 minutes of
stopping the reaction using a microplate reader fitted with a 450nm filter
and with a 620nm to 650nm reference filter. OD values are used to
calculate results.
7- Interpretation of results:
8-1 Warnings
• A negative QuantiFERON®-TB Gold result does not preclude the
possibility of M. tuberculosis infection or tuberculosis disease: false-
negative results can be due to stage of infection (e.g., specimen obtained
prior to the development of cellular immune response), co-morbid
conditions which affect immune functions, incorrect handling of the
blood collection tubes following venipuncture, incorrect performance of
the assay, or other immunological variables.
• While ESAT-6 and CFP-10 are absent from all BCG strains and from
most known non-tuberculous mycobacteria, it is possible that a positive
QuantiFERON®-TB Gold result may be due to infection by M. kansasii,
M. szulgai or M. marinum. If such infections are suspected, alternative
tests should be investigated.
8-2 Precautions
• Green Diluent contains normal mouse serum and casein, which may
trigger allergic responses; avoid contact with skin.
• Deviations from the directions for use in the Package Insert may yield
erroneous results. Please read the instructions carefully before use.
• Do not use kit if any reagent bottle shows signs of damage or leakage
prior to use.
• Do not mix or use ELISA reagents from other QuantiFERON®-TB
Gold kit batches.
• Discard unused reagents and biological samples in accordance with
Local, State, and Federal regulations.
• Do not use the TB peptides and control antigens or ELISA kit after the
expiry date. (60)
BCG Vaccine of Tuberculosis
1: BCG Vaccine
3: Indications
BCG vaccination protects from the sever forms of tuberculosis and has
to be given to the following groups:
• Infants at birth.
• Tuberculin negative contacts of pulmonary smear positive patients.
• Children who are not previously vaccinated.
• Children previously vaccinated but after three month does not
develop BCG scar and remain tuberculin negative.
4: Complications
5- Contraindications
• Fever.
• Pregnancy.
• Children with HIV infection.
• Hereditary immunodeficiency.
• Extensive skin inflammatory conditions and burn.
• Children under immunosuppressive therapy.
• 0.05 ml for neonates and children below one year of age (vaccine
has to diluted in two ml of diluent's and 0.1 ml of the diluted
vaccine is used).
• 0.1 ml for children more than one year of age (the vaccine has to be
diluted in one ml diluent's and 0.1 ml of the diluted vaccine is
used).
• Sterilized syringe with 26 mm has to be used.
8- Site
3- Phases of treatment
5- Duration of treatment
The duration of treatment should be not less than 6 months and there is
no need for expansion of this period if the patient adheres to treatment,
except in some exceptional cases.
The following are the main drugs used in treatment of tuberculosis and
their codes:
• Isoniazid (H)
• Rifampicin (R)
• Pyrazinamide (Z)
• Ethambutol (E)
• Streptomycin (S)
1- Hepatic TB
1-1Clinical:
1-2Diagnosis:
2-2Diagnosis:
The bromide partition test and lactate levels are less useful, but the
polymerase chain reaction (PCR) technique can yield a sensitivity above
*+,# ! - . - / ! 0 ) 1 2 . 3 ! 3 4 - ! !
specificity by decreasing possible laboratory contamination. (93)
Multicenter trials would be most helpful in establishing the clinical
usefulness of these various tests. (94) A meningeal biopsy or brain biopsy
may be necessary, on occasion, but carries significant risk, including
postoperative epidural hematoma and hydrocephalus. (95)
Conclusion
Pulmonary Tuberculosis is a disease caused by germ called
Mycobacterium tuberculosis, its affect primary the lung, and cause
cavitations in the lung, and can affect another sites of body.
The tuberculosis patient must be isolates in hospital for the first two
months from beginning of treatment, and the treatment takes time 6-8
months.
The vaccine give immunity up to 80%, the most active control for this
disease is the healthy education.
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