Mycobacterium tuberculosis
G.Hari Haran
Mycobacteria
• Mycobacteria – fungus like bacteria
• Acid Fast Bacilli (AFB) – They resist decolourisation to acid alcohol
They are
• Aerobic • Non motile • Non sporing• Non capsulated• Obligate parasites
M.tuberculosis complex
• Mycobacterium tuberculosis• Mycobacterium africannum• Mycobacterium bovis
Morphology
• Slender, straight or slightly curved rod• 0.4 x 3 micron in size• Occurs singly, in pairs or as small clusters• Long filamentous club shaped and
branching forms may be seen
Staining Characters
• Acid fast stainingZiehl Neelson stain
Resist decolorisation with acid alcohol
Beaded appearance
Appreciated in oil immersion only
Appear in bright red color
Acid Fast staining
M.Tuberculosis stained red (sputum)
• Fluorochrome stainingPhenolic auramine or auramine rhodamine
used
Increased sensitivity compared to acid fast staining.
Appear as yellow luminous bacilli.
M.Tuberculosis – Fluorescent stained
M.tuberculosis- Electron microscopy
Direct microscopy
• Any biologic fluid or material can also be visualised directly.
• Thin fluids are centrifugated before examination
Culture• Gold standard
• M.tuberculosis grows slowly
• Colonies appear in 4 to 8 weeks
• Optimum temperature is 37 degree centigrade
• Optimum pH 6.4 to 7.0
• Solid and liquid media are available
Commonly used media
• Lowenstein Jensen Medium
• Middlebrook’s
Colony Characteristics
Dry, Rough, Tough, raised and wrinkled, creamy white or Buff colored
Colony Characteristics
Cord growth-Serpentine rods are formed by virulent
strains in liquid media
Biochemical Reactions
Biochemical reaction Result
Niacin test Positive
Aryl sulphatase test Negative
Neutral red test Positive for virulent strains
Catalase Peroxidase test Positive
Amidase test Positive
Nitrate reduction test Positive
Laboratory Diagnosis
Specimens
• Pulmonary TB: Sputum
• Bones and joints: Aspirated fluid. Bone Marrow aspirate
Direct Microscopy
• Grading is done based on number of bacilli seen in Ziehl Neelsen staining
• Examined under oil immersion
0/300 fields AFB not seen
1-2/300 fields Doubtful, repeat smear
1-9/100 fields 1+
1-9/10 fields 2+
1-9/field 3+
10 or more/field
4+
Culture
• Can detect as few as 10 to 100 bacilli per ml
• LJ medium is commonly used
• Smear is prepared from the colony and ZN staining is performed.
Other methods
• BACTEC (Radiometric method) – colonies form in about 1 week.
• Mycobacterial Growth Indicator Tube (MGIT) – Non radiometric automated method.
• Animal inoculation- rarely used these days
Serology
• Enzyme Linked Immuno Sorbent Assay
• Radio Immuno Assay
• Latex aggutination test
Molecular methods
• Polymerase Chain ReactionDNA amplification is done
High sensitivity- 92%
High specificity-99%
Chromotographic Methods
• High Performance Liquid Chromotography
• It is a very specific and rapid method
• Used for species identification
Adenine Deaminase test
• Use in pulmonary TB is controversial
• Useful in TB pleuritis and TB meningitis only
Sensitivity testing• Drug resistant mutants continuously arise.
• Emergence of multidrug resistant TB
• Therefore the tubercle bacilli are tested in LJ media using different concentrations of antitubercular drugs.
Screening methods
• Tuberculin skin testPurified protein derivative is used0.1 ml (5 IU) given ID on flexor aspect of forearmExamined in 48 to 72 hoursPositive reaction- induration of >10mm
surrounded by erythemaFalse negative- miliary tb, LL leprosy, hodgkin’s
diseaseFalse positive- atypical mycobacteria infection
Recent Advances
• Interferon gamma release assays (IGRA)Mycobacterium tuberculosis specific antigens
stimulate release of interferon gamma from T cells.
Lymphocytes from patient’s blood is incubated with the antigens
If the patient was exposed to the TB antigens, IFN gamma will be released from the lymphocytes.
Used to detect latent infection
Thank you