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1. Mycobacterium tuberculosis
Yubraj Bhatta
(M.sc.MLT Clinical Biochemistry)

2. Clinical Manifestations:
Tuberculosis (TB) is classified as:
i) Pulmonary tuberculosis and
ii) Extrapulmonary tuberculosis.
i) Pulmonary Tuberculosis (PTB)
 Pulmonary tuberculosis (PTB) accounts for 80% of all cases of
Tuberculosis (TB).
 They are of two types:
a) Primary tuberculosis
b) Secondary or post primary tuberculosis.

3. a)Primary tuberculosis
• It is the initial infection caused by tubercle
bacilli into a host where the bacilli are
engulfed by alveolar macrophages giving rise
to tuberculosis pneumonia
• Affect the lower lobe or lower part of upper
lobe (ghon focus).
• The hilar lymphnode are also involved. The
ghon focus together with enlarged hilar
lymphnode constitutes the primary complex.

4. b) Secondary tuberculosis:
• Occur due to reactivation of latent infection or
exogenous reinfection.
• Affect mostly upper part of lungs, the lesion undergo
necrosis and tissue destruction leading to cavitations.
• Sign and symptoms are non specific and mainly consist
of fever, night sweat, loss of weight, malaise etc.
• Cough eventually develops with initial non-productive
cough and subsequently accompanied by production of
purulent sputum.
• Extensive disease may lead to respiratory distress
syndrome.

5. ii) Extrapulmonary Tuberculosis (EPTB)
• EPTB results from hematogenous dissemination of tubercle
bacilli to various organs.
• EPTB constitutes about 15-20% of all cases of TB, whereas in
HIV-positive patients, the frequency is much higher
accounting for 20-50% of all cases of tuberculosis.
Cont....

6. Sites commonly involved in EPTB are:
i) Tuberculous lymphadenitis
ii) Pleural tuberculosis
iii) Tuberculosis of upper airway
iv) Genitourinary tuberculosis
v) Skeletal tuberculosis
vi) Tuberculosis of Central nervous system(CNS)
vii)Gastrointestinal tuberculosis
viii)Tuberculosis pericarditis
ix) Tuberculous skin lesions
x) Disseminated tuberculosis (miliary)

7. a) Tuberculous lymphadenitis:
 It is the most common form, accounting for
35% of all EPTB cases.
 The most common on sites are posterior
cervical and supra-clavicular lymph nodes. It
presents as painless swelling in the neck
region
Cont..

8. Cont...EPTB
b) Pleural tuberculosis: It accounts for 20% of all EPTB cases. It
presents as pleural effusion.
c) Tuberculosis of the upper airways- involving larynx, pharynx,
and epiglottis.
d) Genitourinary tuberculosis:
 Renal tuberculosis
In female patients, fallopian tubes and the endometrium are
commonly involved causing infertility.
In males, epididymis is the most common site.

9. Cont..
e) Skeletal tuberculosis:
• Weight-bearing joints eg: spine, hips and knees are
commonly affected.
• With advanced disease, collapse of vertebral bodies results
in kyphosis.
f) Tuberculosis of CNS:
• Seen most commonly in children.
• Tuberculous meningitis is common forms.

10. Cont...
g) Gastrointestinal tuberculosis:
• Terminal ileum and ceacum are the most common sites
involved.
• The route of spread may be due to swallowing of sputum,
hematogenous spread, or ingestion of cow's milk
(unpasteurized) contaminated with M.bovis (in developing
countries).

11. Cont...
h) Tuberculous pericarditis:
• Occurs as direct extension from adjacent lymph nodes or
following hematogenous spread.
• Seen mostly in elderly people, in countries with low TB
prevalence.
i) Tuberculous skin lesions:
• Scrofuloderma: It is a skin condition caused by tuberculous
involvement of the skin by direct extension
• Lupus vulgaris: Apple jelly nodules are formed over the face
in females.

12. Cont..
j) Miliary or disseminated tuberculosis:
• Hematogenous spread of tubercle bacilli results in the
formation of yellowish 1- 2 mm size granulomatous lesions
resembling millet seeds (thus termed as miliary) in various
organs.
• It is more common in HIV-infected people.

13. Cont..
k) HIV-associated Tuberculosis:
• Tuberculosis is one of the most common opportunistic
diseases among HIV infected persons due to low CMI
• Worldwide, TB occurs in 70-80% of HIV-infected
individuals, EPTB being more common than PTB..

14. Laboratory diagnosis:
• Specimen collection:
 In pulmonary Tuberculosis:
Sputum (2 specimens- spot and early morning),
• In Extra pulmonary tuberculosis:
Specimens vary depending on the site involved.

15. Digestion, Decontamination of
Specimen:
• Sputum and specimens from non-sterile sites need prior treatment
for digestion (to liquefy the thick pus cells and homogenization),
decontamination (to inhibit the normal flora) and
concentration (to increase the yield).
• Processing should be carried out in class-II biosafety cabinet.
following methods are available.
i) Petroff's method (4% NaOH/ Sodium hydroxide):
• Sputum is thoroughly mixed with equal volumes of
4% NaOH, centrifuged and the sediment is neutralized with 8%
HCI.

16. Cont..
ii) NALC (N-acetyl-L-cysteine) + 2% NaOH:
• This is superior than Petroff's method for isolation.
• NALC liquefies the sputum and NaOH kills the normal flora.
• This method is more compatible with automated culture
systems.

17. Direct microscopy by Acid-fast Staining
 Ziehl-Neelsen (ZN)Technique
Smears (2 x 3 cm size) are prepared from the mucopurulent part of
sputum or with the sediment obtained after concentration.
• Procedure:
(1) Smear is covered with primary stain, strong carbol fuchsin (basic
fuchsin and phenol mixture) for 5 minutes.
(2) Intermittent heating of the slide is done till the fumes appear.
Heating helps in better penetration of the stain;
(3) Decolorization is done with 20% sulphuric acid for 3 minutes;
(4) Counter staining is done with methylene blue/ malachite green
for 1 minute.
(5) Slide is examined under oil immersion objective.

18. Cont. ZN stain
 Negative result:
• At least 100 oil immersion fields should be examined before
giving a negative report.
 Positive result:
• M. tuberculosis appears as long slender, beaded, less uniformly
stained, red colored acid fast
 Advantages:
• Smear microscopy is rapid, easy to perform and is cheaper.
 Disadvantages:
(1) Smear microscopy is less sensitive than culture as the
detection limit of smear microscopy is 10,000 bacilli/mL of
sputum.
(2) It cannot determine the viability of bacilli.

19. Cont..
 Reporting:
• Microscopy provides only presumptive diagnosis.
• If typical beaded appearance is seen then it should be
reported acid-fast bacilli resembling M. tuberculosis are
seen by smear microscopy by ZN stain.
• It is difficult to differentiate M. tuberculosis from
saprophytic mycobacterium present in tap water or even as
commensals in clinical samples such as gastric aspirate, and
urine.

21. Kinyoun's/ Cold Acid Fast Staining:
• It differs from Z-N staining in that:
1) Heating is not required,
2) Phenol concentration in carbol fuchsin is increased,
3) Duration of carbol fuchsin staining is more.

22. Auramine Phenol Technique
• It is a fluorescence staining technique, uses auramine phenol
as primary stain,
• Acid alcohol as decolorizer and
• Potassium permanganate as counter stain.
• The bacilli appear bright green against dark background
• Smears are screened by using 20 X objective, hence can be
screened faster (2 min for 1OO fields).
• It is widely used by RNTCP( Reserved national tuberculosis
control programme) in laboratories having higher sample
load.

23. Culture Techniques:
• Culture Conditions
 Incubating condition: Tubercle bacilli are slow growing due to
long generation time (10-15 hours).
 Hence, inoculated culture media are incubated at 37'C, for 6-8
weeks.
 Media are incubated either aerobically for M. tuberculosis
 Microaerophilic condition (5% 02) for M. bovis.

24. Culture Media:
Egg-based media: Examples include
i) Lowenstein-Jensen (LJ): It consists of coagulated hen's eggs,
mineral salt solution. asparagine and malachite green
Colony appear as rough, tough and buff colored.
ii) Petragnani and
iii) Dorset egg media:
Other solid media:
• Blood-based (Tarshis mediun)
• Serum-based (Loeffler mediun)
• Potato-based (Pawlowsky medium)
• Agar-based (Middlebrook 7H10 and 7H11)

25. Liquid media:
• Not usually used for culture instead it is used for drug
sensitivity testing and preparation of antigen and vaccine.
• E.g:
i) Middlebrook 7H9,
ii) Dubos,
iii) Proskauer,
iv) Sula, and Sauton media.
Advantage of culture:
Culture is more sensitive than microscopy as it has a detection
limit of just 10- 100 viable bacilli.

26. Automated culture methods:
• Several automated culture methods have been developed in last
decade.
• They are rapidly gaining popularity.
• Many hospitals, nowadays prefer to use these systems as they have
many advantages over the conventional LJ culture.
 BACTEC MGIT (Mycobacteria growth indicator tube):
 Uses an oxygen sensitive fluorescent compound, dissolved in the
broth. Initially,
 The large amount of dissolved oxygen quenches emissions from the
compound and little fluorescence can be detected.
 Later, actively respiring microorganisms consume the oxygen and
the absence of oxygen allows the fluorescence to be detected

27. Cont..
BAcT/Alert:
• Uses the principle of colorimetric detection of pH change in
the medium, which occurs due to CO2 liberated by the growth
of M. tuberculosis
ESP system:
• Detects a change of pressure in the medium which occurs
due to the production of CO2 by M. tuberculosis
BACTEC 460:
• It was the first semi automated system developed. As it was
based on the use of radioisotopes to detect growth, hence
not in use currently

28. Biochemical Identification:
• Colony smears from LJ bottles are stained to demonstrate acid
fast bacilli. Various biochemical tests are done to differentiate
M. tuberculosis from other mycobateria.
 Tests to Differentiate M. tuberculosis from M. bovis:
1) Niacin test:
Positive for M. tuberculosis However, this is negative for M.
bovis.
2) Nitrate reduction test:
Positive for M. tuberculosis and negative for M. bovis.
3) Pyrazinamidase test: M. tuberculosis produces pyrazinamidase,
therefore, hydrolyses pyrazinamide. This test is negative for M.
bovis.
4) TCH (Thiophene caroboxylic acid hydrazide): M. tuberculosis is
resistant to TCH, whereas M. bovis is sensitive.

29. Tests to Differentiate Tubercle Bacilli from NTM
 Catalase test:
• Tubercle bacilli (human and bovine) are weakly catalase positive
where as
• Non-tuberculous mycobacreria (NTM) are strongly catalase
positive.
NOTE: Isoniazid resistant strains lose their catalase activities.
 Arylsulfatase test:
• Positive for rapid grower group of NTM, but negative for tubercle
bacilli.
 Tween 80 hydrolysis test:
• Positive for certain NTM, such as M. kansasii and M.marinun.
M. tuberculosis shows variable results.

30. Serology:
 Antigen detection:
 Various formats such as capture ELISA, dip stick and latex
agglutination tests are available for detection of specific M.
tuberculosis antigens such as lipoarabinomannan and
antigen-5 in sputum, urine and other samples.
 These tests are less popular because their sensitivity is low
(40-50%).
 • Antibody detection:
 Methods are no longer recommended in endemic areas
because of cross reactivity with other environmental
mycobacteria and variable antibody response is seen.

31. Molecular Methods
 Advantages:
• They take less time than culture
• They are more sensitive than culture, can detect as low as 1
bacillus/mL of specimens. This is very much useful for
extrapulmonary samples.
• They can also detect the drug resistance genes.
• Used for epidemiological typing of scrairis.

32. Molecular methods available are:
 Gene expert:
Identification of strains of Mycobacterium tuberculosis which are
resistant to rifampicin drug can be identified by nucleic acid testing.
Other methods include:
• Probe based identification (Line probe assay)
• Transcription mediated amplification (TMA)
• Strand displacement amplification (SDA)
• Nucleic acid sequence based amplification (NASBA)
• Ligase chain reaction (LCR).

33. Animal pathogenicity Testing
 Guinea pig pathogenicity test
 Both M.tuberculosis and M. bovis are pathogenic to guinea pigs.
 Intramuscular inoculation of the concentrated specimen produces
progressive weight loss of the animal with positive tuberculin test
(after 3-4 weeks) and death (after 6 weeks).
 Autopsy of the animal shows:
• Caseous lesion at the site of inoculation.
• Enlarged caseous inguinal lymph nodes.
• Tubercles may be seen in spleen, lungs, liver or peritoneum, but
kidneys are unaffected.

34. Cont..
Rabbit pathogenicity test:
M. bovis is pathogenic, where as M.
tuberculosis is not pathogenic to rabbit.

35. Diagnosis of EPTB
Diagnosis of EPTB differs from PTB in the following aspects:
• The EPTB specimens are paucibacillary (bacilli present in few
numbers) hence smear microscopy is less sensitive. therefore,
molecular methods are more useful in the diagnosis of EPTB.
• Specimens are free of normal flora; hence, initial
decontamination and concentration methods are not required.
• Pleural fluid examination reveals elevated ADA (adenosine
deaminase) and interferon (IFN)-gamma levels.

36. Cont..
• ln renal tuberculosis: Urinary excretion of bacilli is
intermittent; hence 3-6 consecutive early morning urine
samples are collected, centrifuged and the sediment is used
for processing. Acid alcohol is used as decolourizer.
• In tuberculous meningitis: CSF examination shows cobweb
coagulum on standing, elevated CSF pressure, raised protein
and chloride levels; whereas glucose levels is decreased.

37. Typing of Mycobacteria
• Is useful for epidemiological studies in determining the
relatedness between various isolates in the communities.

38. Diagnosis of Latent Tuberculosis
• Latent tuberculosis is diagnosed by demonstration of delayed
or type IV hypersensitivity reaction against the tubercle
bacilli antigens.
• Two methods are available,
i) Tuberculin test,
ii) IFN- gamma release assay

39. Tuberculin test
• Procedure:
Mantoux test: It is the most commonly employed mediod.
0.1 mL of PPD containing one TU(tuberculin unit) is
injected intradermally into fexor surface of forearm.
• Reading:
It is taken after 48-72 hours. At the site of inoculation, an
induration surrounded by erythema is produced.
 If the width of the induration is:
i) More than 0r equals to 10 mm: Positive (tuberculin
reactors)
ii) 6-9 mm: Equivocal/doubtful reaction
iii) <5 mm: Negative reaction

41. Cont.. Tuberculin test
• Interpretation of result:
 Adults: Positive tuberculin test in adults only indicates
present or past exposure with tubercle bacilli but does not
confirm the presence of active stage of the disease. Hence it is
only used as an epidemiological marker.
 Children: In children, positive test indicates active infection
and used as diagnostic marker.

42. Cont..
False-positive:
The test becomes positive after:
• BCG vaccination (after 8- 14 weeks)
• Nontuberculous mycobacteria infection
False-negative:
The test may become negative in various conditions such as-
• early or advanced TB,
• miliary TB,
• decreased immunity

43. Interferon Gamma Release Assay(IGRA)
• This uses highly specific M. tuberculosis antigens such as
CFP10 (culture filterate protein) and ESAT6 (early secreted
antigenic target-6); both coded by RD1 genes.
• Procedure:
It is an in-vitro test. Sensitized T lymphocytes collected from
suspected individuals, are exposed to ESAT-6/CFP-IO
antigens, which leads to release of high level of IFN-gamma
from the T lymphocytes. An ELISA formats is available
commercially (QuantiFERON-TB Gold assay).
• Advantage:
It is highly specific, there are no false positive conditions.

44. Anti-microbial sensitivity testing:
• Commonly used AST include the following:
i) Absolute concentration method
ii) Resistance ratio method
iii) E- test
iv) Nitrate reduction test

45. Prophylaxis:
• Classified as following:
i) General prophylaxis
ii) Immuno prophylaxis
iii) Chemo prophylaxis
 General prophylaxis:
• Vaccination of new born with BCG vaccine.
Providing tuberculosis awareness programme to public
• Early diagnosis of tuberculosis and their treatment.
Pasteurization of milk.

46. Immuno prophylaxis:
• BCG vaccine ( prepared from live attenuated strain of M.bovis
• 0.1 ml is given intradermally as soon as birth and if missed
before one year of birth.
Complication:
Prolonged ulceration at the site of vaccination.
Lymphadenitis
Osteomyelitis
cont...........

47. Cont... Immuno prophylaxis
• Contraindication
BCG should not be given to patient suffering from following
infection..
i) Eczema
ii) HIV
iii) Leukemia
iv) Lymphoma
v) Patient under immunosupressive treatment.

48. Treatment (chemopropylaxis)
Anti-tubercular drugs can be
cassified into:
• First line drugs
i) Isoniazid (H)
ii) Rifampicin (R)
iii) Pyrazinamide (Z)
iv) Ethambutol (E)
v) Streptomycin (S)
• Second line drugs
i) Ethionamide
ii) Quinolones  Ofloxacin
and ciprofloxacin
iii) Aminoglycosides 
Kenamycin, capremysin
and amikacin
iv) Cycloserine
v) Macrolides

49. DOTS:
• DOTS is recommend by RNTCP (Revised national tuberculosis
control programme) and WHO (World Health Programme).
Here, the strategies used are:
i)The entire treatment course is supervised to improve the
patient's compliance.
ii) Treatment response is also monitored by periodic sputum
smear microscopy.

50. References:

51. Thank you 