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Important Points

  1. Straight or slightly curved rods; Filamentous or branching forms may occur
  2. Mould like pellicle in liquid cultures therefore called mycobacterium
  3. Gram positive but stain poorly, acid fast (20%); mycolic acid + integrity of cell wall
  4. ZN; Kinyoun; Fluorochrome staining (Auramine & Rhodamine; most sensitive staining method)
  5. Aerobic or microaerophilic, nonmotile, nonsporing 

A. Mycobacterium tuberculosis complex

  1. M tuberculosis
  2. M bovis
  3. M bovis BCG
  4. M africanum
  5. M microti
  6. Recent additions to M. TB complex- M. canetii, M. caprae, M. pinnipedi 

B. Diagnosis:

  1. Microscopy: > 10,000 bacilli/ ml; 20-80%
  2. Culture: can detect: 10-100 bacilli/ ml 

C. Culture:

  • M tuberculosis: obligate aerobe and M bovis: microaerophilic

1. Solid media:

  1. L J; Petragnini; Dorset, American thoracic society medium : egg based
  2. Tarshis: blood based
  3. LSS: serum based
  4. Pawlowsky: potato based
  5. Middlebrook 7H11 and 7H10-agar based

2. Liquid media:

  1. Dubo’s, Middlebrook’s, Sula & Sauton
  2. Cord factor in virulent strains: serpentine cords in liquid media 

D. Newer culture methods : - (Recent Advances):

  1. BACTEC: C 14 labelled palmitic acid
  2. PANTA (polymixin, amphotericin B, nalidixic acid, trimethoprim, azlocillin)
  3. Growth index >50 considered positive
  4. NAP (paranitro-α-acetylamino-βhydroxy-propiophenone) containing media: growth rules out MTB complex
  5. (Mycobacterium growth indicator tube system) MGIT
  6. 7H9 broth with fluorescent compound in silicone sensor. Oxygen diminishes/quenches the fluorescent output of the sensor. Oxygen consumption leads to increase in fluorescence
  7. SeptiChek AFB system-manual detection method
  8. Biphasic culture system (7H9 broth and three sided paddle [chocolate, egg-based & 7H11 agar])
  9. ESP culture system-7H11 broth, cellulose sponge
  10. Pressure changes due to oxygen production or gas production detected
  11. BacT/Alert-an automated colormetric method which uses a gas permeable sensor that changes color when Co2 is generated by growing bacteria.
  12. Luciferase reporter phage system
  13. Molecular methods (most sensitive): PCR (target amplifying IS 6110); Ligase chain reaction (LCR); Transcription mediated amplification (TMA)
  14. DNA probes: can distinguish between species 

E. Sensitivity testing:

  1. Absolute concentration (MIC)
  2. Resistance ratio-Test strain & known standard strain (H37Rv)
  3. Lowest concentration showing 20 colonies taken as end point
  4. Ratio of MIC of test strain to MIC of known strain
  5. Ratio of 8: resistant and Ratio of 2: sensitive
  6. 1% proportion method-WHO recommended
  7. Standard inoculum of drug containing & drug free medium
  8. Resistant: more than 1% bacteria grow in the presence of the drug
  9. Radiometric automated systems- Based on 1% proportion method
  10. Phage based assays and MODS
  11. Molecular methods for detection of mutations associated with resistance 

F. Drug resistance in tuberculosis:

  1. MDR-TB: Resistance to INH & Rifampicin with/ without resistance to other drugs
  2. XDR-TB: Strain of M. tuberculosis resistant to INH, Rifampicin and three or more than three classes of second line drugs
  3. Recently XDR is changed to MDR + resistance to any fluoroquinolone and one of the 3 injectable drugs used in ATT, i.e,Capreomycin, Kanamycin and Amikacin.
  4. Primary drug resistance: patient who have never taken drug in the past & get infected with strain originating from patients who had acquired resistance
  5. Acquired resistance: Resistance acquired after intake of drug by patient
  6. Selection of pre-existing mutant because of inaccurate chemotherapy
  7. Natural resistance: Neither the patient with naturally resistant bacilli nor his source of infection has had chemotherapy in the past
    1. Spontaneous mutations                            
    2. Single drug
    3. INH & Rcin: 1/108 - 109 replications               
    4. SM: 1/106 replications
    5. Ethionamide, cycloserine, Thiacetazone: 1/103 replications
INH kat G
inh A
ahp C
Rcin rpo B
SM rps L, rrs, str A, S12
EM emb A, B, C
Pzide pnc A
Flouroquinolones gyr A, B

CHARACTER

CLASSIC HUMAN

CLASSIC BOVINE

Morphology

Straight, slightly curved, singly or in pairs or small clumps;

Beaded or barred forms are seen

Straighter, stouter, shorter

Uniformly staining

Growth requirement

Obligate aerobe

Microaerophilic

5-10% CO2

Growth enhanced

Not enhanced

Glycerol at 0.75%

Stimulates

Inhibits

Glycerol at 0.5%

Stimulates

Stimulates

Pyruvate enhancement

Yes

Yes

Type of growth

Eugonic

Dysgonic

Appearance

Rough, tough, buff

Smooth, flat, moist

Pzide sensitivity

Yes

No

Tween 80 hydrolysis

Yes

No

TCH sensitivity

No

Yes

Niacin

+

-

Nitrate

+

-

Phage type

A,B,C

A

 





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