2. Mycobacteia are slender rods that sometimes show branching,
filamentous forms resembling fungal mycelium
Classification
The genus Mycobacterium contains three groups
1.Obligate parasites
2.Opportinistic pathogens
3.Saprophytes
4. Opportunistic pathogens
Non-tuberculous mycobacteria (NTM)
This group contains mixed group of isolates from diverse
sources: birds, cold-blooded and warm-blooded animals,
from skin ulcers, and from soil, water and other
environmental sources
They are opportunistic pathogens and can cause many types
of disease
5. Mycobacterium tuberculosis
• long, slender, straight or curved,
about (3 x 0.3 µm in size)
• Aerobe
• Acid fast bacilli
• Intracellular
• Mycolic acid, waxes & lipids
in cell wall
• Slow growing
(Doubling time: 15 – 20 hours)
6. The Nobel Prize in Physiology or Medicine
1905
In 1882 while working in Berlin
he discovered the tuberculosis bacteria
and the means of culturing it
7. Pathogenesis
Source of infection
Open case of pulmonary tuberculosis
Mode of infection
Direct inhalation of aerosolised bacilli contained in the
droplet nuclei of expectorated sputum
Infection also occurs infrequently by ingestion for example,
through infected milk, and rarely by inoculation
8. Transmission of M. tuberculosis
Millions of tubercle bacilli in lungs (mainly in
cavities)
Coughing projects droplet nuclei into the air
that contain tubercle bacilli
• One cough can release 3,000
droplet nuclei
• One sneeze can release tens
of thousands of droplet
nuclei
9. M. tuberculosis does not spread by:
• Sharing dishes and utensils
• Using towels and linens
• Handling food
• Sharing cell phones
• Touching computer keyboard
10. The initial infection with M. tuberculosis is referred to as a
primary infection
Subsequent disease in a previously sensitized person, either
from an exogenous source or by reactivation of a primary
infection is known as postprimary tuberculosis
Both forms exhibit quite different pathological features
11. Primary tuberculosis
It is the initial infection by tubercle bacilli in a host
The site of the initial infection is usually the lung
These bacilli engulfed by alveolar macrophages, multiply
and give rise to a subpleural focus of tuberculous
pneumonia
Which is commonly located in the lower lobe or lower part
of the upper lobe to form the initial lesion or Ghon focus
Some bacilli are carried to the hilar lymphnodes through
macrophages, where additional foci of infection develops
12. The Ghon focus, together with the enlarged hilar
lymphnodes, form the primary complex
M. tuberculosis multiply within the alveolar macrophages
Th-1 cells produce cytokines to activate these macrophages
Activated macrophages effectively destroy most of the
tubercle bacilli
However, some bacilli escape the macrophage- mediated
destruction and induce the hypersensitivity reaction
A hard tubercle or granuloma is formed due to the
hypersensitivity reaction
13. When fully developed, tubercle/granuloma consists of 3 zones
1. A central area of large, multinucleated giant cells containing
tubercle bacilli
2. A mid zone of pale epitheloid cells, often arranged radially
3. A peripheral zone of fibroblasts, lymphocytes and
monocytes
Later, peripheral fibrous tissue develops, and the central
area undergoes caseation necrosis
A caseous tubercle may break into a bronchus, empty its
contents there, and form a cavity
It may subsequently heal by fibrosis or calcification
16. Postprimary (secondary) tuberculosis
It is due to reactivation of latent infection or exogenous
reinfection and differs from the primary type in many respects
It is characterised by chronic tissue lesions, the formation of
tubercles, caseation and fibrosis
Regional lymphnodes are only slightly involved, and they
do not caseate
Postprimary tuberculosis always begins at the apex of the lung,
where the oxygen tension is highest
The necrotic materials break out into the airways, leading to
expectoration of bacteria-laden sputum, which is the main source
of infection to contacts
17. Differences beween primary and postprimary
tuberculosis
Characteristics
Site
Local lesion
Cavity formation
Lymphatic
involvement
Infectivity*
Local spread
*Pulmonary cases
Primary
Postprimary
Any part of lung Apical region
Small
Large
Rare
Frequent
Yes
Minimal
Uncommon
Uncommon
Usual
Frequent
18. Immunology
Tubercle bacilli do not contain or secrete a toxin
The exact basis of their virulence is not understood, but
seems to be related to their ability to survive and multiply
in macrophages
Humoral immunity appears to be irrelevant
The only specific immune mechanism effective is the CMI
19. The key cell is the activated CD4+ helper T cell which can
develop along two different paths: The Th1 and Th2 cells
Th1 dependent cytokines activate macrophages, resulting
in protective immunity and containment of the infection
Th2 cytokines induce delayed type hypersensitivity (DTH),
tissue destruction and progressive disease
20. Koch’s phenomenon
Koch’s phenomenon is a combination of hypersensitivity
and immunity
It is the response of a tuberculous animal to reinfection
When a healthy guinea pig is inoculated subcutaneously
with virulent tubercle bacilli, the puncture site heals quickly
After 10-14 days, a nodule appears at the site of injection
which ulcerates and the ulcer persists till the animal dies of
progressive tuberculosis
21. If on the other hand, virulent tubercle bacilli are injected in a
guinea pig, which had received a prior injection of tubercle bacilli
4-6 weeks earlier, an indurated lesion appears at the site of injection
in a day or two which undergoes necrosis to form a shallow ulcer
This ulcer heals rapidly without involvement of the regional
lymphnodes or tissues. This is called Koch’s phenomenon
Koch’s phenomenon has got three components
1. A local reaction of induration and necrosis
2. A focal response in which there occurs acute congestion
and even hemorrhage around the tuberculous foci in tissues
3. A systemic response of fever that may sometimes be fatal
22. Laboratory diagnosis
Specimen collection
Type of lesion
Specimen
Pulmonary tuberculosis
Sputum
Laryngeal swabs or
bronchial washings
Gastric lavage
Renal tuberculosis
Urine
Tuberculous meningitis
CSF
Early morning sputum samples should be collected for 3
consecutive days in a sterile container
In case of renal tuberculosis, 3-6 morning urine samples
should be collected
23. Concentration of specimens
Concentration of a specimen is done to achieve;
1. Homogenisation of the specimen
2. Decontamination i.e. to kill commensal bacteria
3. Concentrate the bacilli in the specimen without inactivation
The concentrate is used for smear preparation, cultutre and
animal inoculation
Petroff’s method is used to concentrate sputum specimens
26. Direct Microscopy
Ziehl-Neelsen staining
(hot staining method)
Kinyoun’s method
(cold staining method)
Acid fast bacilli resist decolourisation with acid and alcohol
once they have been stained with carbolfuchsin
AFB appear as pink, long, slender bacilli with beaded
appearance
27. Fluorescent staining by Auramine O or
auramine rhodamine
Mycobacterium spp. will
fluoresce yellow against
dark background under
fluorescent microscope
28. Diagnosis of pulmonary tuberculosis
under RNTCP
DOTS: Directly observed treatment short-course
29. Culture
Concentrated specimen is inoculated
on Lowenstein – Jensen’s medium and
incubated at 370C for 2 – 8 weeks
M. tuberculosis
Colonies appear as buff coloured, dry,
irregular colonies with wrinkled surface
and not easily emulsifiable
(Buff, rough and tough colonies)
Colonies are creamy white to yellow colour
with smooth surface and easily emulsifiable
M. bovis
31. Biochemical reactions
Niacin test
M. tuberculosis lacks the enzyme that converts Niacin to
Niacin ribonucleotide due to this large amount of Niacin
accumulates in the culture medium
Niacin is detected by addition of
10% cyanogen bromide and
4% aniline in 96% ethanol
Positive reaction – canary yellow
M. tuberculosis – Positive
M. bovis - Negative
32. Nitrate reduction test
M. tuberculosis produce an enzyme nitro reductase which
reduces nitrate to nitrite
This detected by colorimetric reaction
by addition of sulphanilamide
and n-naphthyl- ethylene diamine
dihydrochloride
Positive reaction – pink or red colour
M. tuberculosis – Positive
M. bovis - Negative
33. M. tuberculosis is resistant to TCH (Thiophene - 2
- carboxylic acid hydrazide); hence, growth occurs
Growth in presence of TCH
M. bovis is susceptible; therefore,
does not grow
M. tuberculosis
M. bovis
34. Rapid culture methods
1. BACTEC
2. Mycobacterial growth indicator tube (MGIT)
3. Bac T/ Alert 3D system
BACTEC system
Average time to detect Mycobacterium
growth is 8 days
Radio metric method
Detects the presence of Mycobacteria
based on their metabolism rather than
visible growth
35. 0.5 ml of processed sample is added to 4 ml of Middlebrook
7H12 broth containing C14 radio labelled palmitic acid
Mycobacteria metabolises C14 radio labelled palmitic acid and
release radio actively labeled 14CO2
BACTEC 460 instrument measures
14CO and reports in terms of growth
2
index (GI)
A growth index of 10 or more is
considered positive
More sensitive than traditional method
Problem of disposal of radio active
waste
36. Animal inoculation
0.5 ml of concentrated specimen is
inoculated intramuscularly into the
thigh of two healthy guineapigs
The animals are weighed prior to
inoculation and thereafter at
weekly intervals
Tuberculin test is done after 3 – 4 weeks
Progressive loss of weight and positive tuberculin skin
reaction indicates infection
One animal is killed after 4 weeks and autopsied, if it shows
no evidence of tuberculosis the other animal is autopsied
after 8 weeks
37. Autopsy shows
1. Caseous lesion at the site of inoculation
2. Enlarged caseous inguinal lymph nodes
3. Tubercles may be seen in spleen, lungs, liver, or peritoneum
4. Kidneys are not affected
38. Allergic tests
Tuberculosis infection leads to the development of delayed
hypersensitivity to M. tuberculosis antigen, which can be
detected by Mantoux test
Mantoux test (tuberculin test)
0.5 ml of PPD containing 5 TU is
injected intradermally on flexor
aspect of fore arm
39. Site is examined after 48 – 72 hrs
Induration of 10 mm or more is
considered positive
Positive tuberculin test indicates
hypersensitivity to tuberculoprotein
denoting infection with tuercule bacilli
or BCG immunisation, recent or past
with or without clinical disease
40. Uses
1. To diagnose active infection in infants and young children
2. To measure the prevalence of infection in community
3. Indication of successful BCG vaccination
41. Detection of antibodies
Various methods such as enzyme linked immunosorbent assay
(ELISA), radio immunoassay (RIA), latex agglutination assay
have been employed for detection of antibodies in
patient serum
However, diagnostic utility of these methods is doubtful
WHO has recommended that these tests should not be used
for diagnosis of active tuberculosis
42. Quantiferon-Gold
Is an in vitro assay that measures the cell mediated immune
-response in the infected individuals through the levels of
interferon gamma (IFN-γ) released by the sensitised
T- lymphocytes after stimulation by M. tuberculosis antigens
43. Molecular methods
1. Polymerase chain reaction (PCR)
2. LAMP
3. Ligase chain reaction
PCR
Rapid method to detect M. tuberculosis directly in clinical
samples based on DNA amplification
IS6110 sequence is generally targeted for detection
M. tuberculosis complex
44. Prophylaxis
General measures
Adequate nutrition, good housing and health education are as
important as specific antibacterial measures
Immunoprophylaxis
The BCG (Bacille Calmette-Guerin) vaccine (0.1 ml), administered
soon after birth by intradermal Injection failing which it may be
given at any time during the first year of life
This is a strain of M. bovis attenuated by 239 serial subcultures
in a glycerine-bile-potato medium over a period of 13 years
46. Chemoprophylaxis
This is the administration of antituberculous drugs
(usually only isoniazid)
1. To persons with latent tuberculosis (asymptomatic tuberculin
positive)
2. To persons with a high risk of developing active tuberculosis
3. To the infant whose mother with active tuberculosis
4. To the children living with a case of active tuberculosis in the
house
Isoniazid 5 mg/kg daily for 6 – 12 months is the usual course
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