Hari TB Microbiology

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