Mycobacteria

Objectives
1. To know the general characteristics of mycobacteria, and
differentiate them from other groups of organisms
2. To be a ware of the safety precautions while working in a
mycobacteriology laboratory
3. To know appropriate specimen collection and processing
procedures to recover mycobacteria from clinical samples
4. To know the principle and procedures for stains used to
demonstrate mycobacteria in clinical samples isolates
5. To know different culture media used for isolation of
mycobacteria
6. To the different tests used to identify mycobacteria
7. To be familiar with the pathogenesis and the clinical disease
caused by mycobacterium tuberculosis
8. To know the clinical significance of nontuberclosis mycobacyteria
9. To be familiar with treatment with anti-TB drugs
 Mycobacteria
1. Mycobacterium tuberculosis complex

• M. tuberculosis
• M. bovis
• M. africanum

2. Non-tuberculosis mycobacteria (NTM) or Atypical mycobacteria

A- Slow –Growing

• Nonphotochromogens:
• Photochromogens
• Scotochromogenes

B- Rapid –Growing

C- Non-cultiveable

• M. leprae
 MYCOBACTERIUM TUBERCULOSIS
MORPHOLOGY

• Thin, straight or curved rods, obligate aerobes that do not

form spores
• Single, pairs or in masses
• Cell wall has high lipid content (60%) mycolic acid

Other lipids include

Mycosides,
Sulfolipids
 Lipoarabinomannan (LAM) extending from plasma memmbrabe
to the surface

• Cell wall not stained by gram staining

• Stained with Ziehl-Neelsen Staining

• Once stained with red dye, resist decolorization with 3% HCl in

alcohol - Acid Fast Bacilli (AFB)

• Appear red against blue background

 RESISTANCE
• Resist dryness
• Survives in dried expectorated sputum
• Resist disinfectants
• Resist acids : basis for staining
• Resist alkalis
• NaOH is used to treat clinical specimens to destroy
unwanted bacteria, human cells, mucous; NOT M.
tuberculosis.
• Heat sensitive: killed by pasteurization
 VIRULENCE FACTORS
• M. tuberculosis does not produce classical
endotoxins or exotoxins

• Virulent strains demonstrate cording in which

organisms remain attached in parallel bundles to
form long interwining cords or ropes

Disease process is largely due to:

• Survival and multiplication in macrophages

• Delayed hypersensitivity reaction to

tuberculoproteins

• Cell wall lipids : produce granulomatous lesion

 SITES OF INFECTION

• Lungs – Pulmonary TB

• Genitourinary tract
• Lymph nodes
• Bones and Joints Pott's disease
(tuberculosis spondylitis )
• GIT Extra-pulmonary TB
• Meninges
• Skin
 TUBERCULOSIS

CAUSATIVE ORGANISMS
° M. tuberculosis 96-97%
° M. bovis 2-3%
° Atypical Mycobacteria 2%

• 1/3rd of world population is infected

with M. tuberculosis
• 8 million new cases per year
• 3 million deaths per year due to TB
 Mode of Transmission of Pulmonary TB

• Respiratory droplet – coughing, sneezing

• One cough may contain 3000 infected
droplet
• Only 10 bacilli may initiate pulmonary
infection in susceptible individual

Factors
° Overcrowding (close contact)
° Frequency of cough
° Number of bacilli in sputum
 PATHOGENESIS OF TUBERCULOSIS
TYPES
• Primary infection
• Reactivation TB

PRIMARY INFECTION (usually in children)

• First exposure to M. tuberculosis

• Dissemination to other organs through blood stream

can occur

• Cell mediated immunity & Hypersensitivity

o Leads to granuloma (tubercle) formation in 2-6 weeks
PATHOGENESIS OF PRI. TB
Lymph
node

MTB Blood
LUNGS
subpleural area
of mid zone
Ingested by Survive & multiply Presented to
MTB inhaled macrophages in macrophages T- lymphocytes

Different
Organs
Limit bacterial Produce lymphokines (Miliary TB)
multiplication attract monocytes to
site of infection

GRANULOMA
(Microscopic)
Monocytes transform to
activated macrophages
(Epitheloid cells)
Granuloma formed at
Pri. Infection site is
Macrophage fuse together to
form multinucleated giant cells
Known as Ghon focus
 PATHOGENESIS OF TB

Multinucleated
giant cells Central
Lymphocytes Epitheloid cells caseation

GRANULOMA WITH
GRANULOMA CENTRAL CASEATION

• When many bacteria cause infection →high degree of hypersensitivity

• Enzymes and reactive oxygen intermediates are released

by dying macrophages →

• Cause caseation necrosis of center of granuloma

OUTCOME OF PRIMARY TB INFECTION

A. Bacterial multiplication stops

Organisms die, lesion in the lung and LN calcify
Classical picture on X-Ray – Ghon Complex.

B. Active pulmonary TB (less common)

C. Bacteria remain viable for long periods

May reactivate after months or years.

D. Disseminated TB (miliary TB). Due to:

Opening of a tubercle into bronchus
Erosion of a blood vessel (blood stained sputum)
 PATHOGENESIS OF TUBERCULOSIS

REACTIVATION TB

1.Occur due to reactivation of an old focus in

elderly due to:

• Malnutrition
• Diabetes mellitus
• Alcoholism
• Immunosuppression (AIDS)

2.Inhalation of new bacilli in person with primary

infection
Diagnosis Clinical Features o
Pulmonary TB
• Low grade fever (evening rise of temp)

• Night sweats

• Fatigue

• Anorexia

• Weight loss

• Productive cough (Blood stained sputum)

Clinical features of Extra-pulmonary TB

According to the organ affected

Bone TB: e.g. Caries spine

Lymph nodes TB: discharging

sinuses in neck
 TUBERCULIN (MONTOUX) TEST

• Purified Protein Derivative (PPD) from

tubercle bacilli
(standardized in Tuberculin Units - TU) is
injected intradermaly

DOSE
• 5TU is usual dose – if test negative;
increase dose to 250 TU
• The test is read after 48 – 72 hours
 INTERPRETATION OF TUBERCULIN TEST

POSITIVE TEST

• Induration of ≥ 10 mm with erythema

• Induration of 5-9 mm – low level sensitization with
tubercle bacilli or cross reacting NTM
• In AIDS patients: 5 mm induration – positive test

Indicates
• Active disease
• Infection by M. tuberculosis at sometime in life
• Previous vaccination with BCG
• Infection with strongly cross reacting NTM
• Child < 5 years if not vaccinated: active disease
 INTERPRETATION OF TUBERCULIN TEST

NEGATIVE TEST
• No induration or < 5 mm
• Not infected with MTB
• Pre-hypersensitivity stage of primary infection

False negative test

° Early TB (test becomes positive after 4-6 weeks
of infection)
° Miliary TB
° Immunosuppression (AIDS)
 Laboratory Diagnosis
Mycobacteria are detected in direct smears of clinical material
contaminated with normal flora must be treated by
mucolytic agents in the sputum to dissolve mucus (N- using ]1[
(acetylcysteine

decontaminating and a digesting agent Combined with NaOH as a ]2[

Cultural media
• Egg based media: LJ contain fresh whole eggs, glycerol,
potato flour and malachite green, positive cultures are detected
in 4-6 weeks
b)Serum or Agar. –positive cultures are detected in 3-4 weeks

c) Liquid Media: The use of liquid media system reduces the isolation
to 10 days compared with 3-6 weeks solid media.

BACTEC system which is automated radiometric culture system. It

contain a14C- labelled substrate that is metabolied by MB
liberating radioactive CO2. The amount of14CO2 liberated is
detected by BACTEC system
 LAB IDENTIFICATION OF TB

SPECIMENS

PULMONARY TB
• First morning sputum for 3 successive days
• Bronchial washings after bronchoscopy
• Bronchoalveolar lavage (BAL)
• Gastric washings (especially in children)
• Pleural fluid in Pleural Effusion

EXTRA-PULMONARY TB
° First morning whole urine for 3 successive days
° Pus or tissue (whole LN) rather than swab
° Liver and bone marrow biopsy in disseminated TB
° CSF in meningitis
 LAB IDENTIFICATION OF TB
EXAMINATION OF STAINED SMEAR

• Direct specimen smear or sediment of urine, CSF, gastric

washing is stained with:

° Ziehl-Neelsen method

° Auramine-Rhodamine fluorescent stains

° Positive film is diagnostic

° Negative film does not exclude TB

° 65% of culture +ve sputum samples yield +ve smear

° A clean voided urine by male patient may be contaminated with M smegmatis

from prepuce

° Bronchoscopes may get contaminated with free-living mycobacteria

 LAB IDENTIFICATION OF TB
CULTURE
• Sediment of Bladder urine, CSF, pleural fluid
cultured directly
• Sputum & urine sediment treated with
2% NaOH to kill contaminants
Inoculated onto:
• Lowenstein Jensen Medium – growth in 4-6 weeks
• Middlebrook Agar (7H11) – growth 2-3 weeks
• Identification of growth by
• Colonial morphology
• pigment, time of growth
• Biochemical tests e.g. M. tuberculosis produce niacin
and others do not
 ZIEHL-NEELSEN STAINING
COLD vs HOT METHODS

HOT Methods

• Heating of carbol fuchsin is recommended to

ensure good staining

COLD Methods (Kinyuoun)

• The concentration of carbol fuchsin &

phenol is increased
• A wetting agent -Tween 80 (that lowers surface
tension of a liquid) is added to ensure rapid
penetration of stain.
 LAB IDENTIFICATION OF TB

Bactec System

• Middlebrook broth (7H9) – radiometric assay

• Detection in 1-2 weeks
° 7H9 medium contains 14C – labeled palmitic acid
° Metabolized by mycobacteria to liberate 14CO2
° Detected automatically by a senosor

NUCLEIC ACID STUDIES

• Polymerase chain Reaction (PCR)
• Nucleic Acid Probes - labeled Mycobacterial DNA is
hybridized with rRNA of test strain
 LAB IDENTIFICATION OF TB

• AFB staining
• Fluorescent staining
• Culture
o On LJ medium
o Bactec System (automation)
• PCR
 ZIEHL-NEELSEN STAINING

Used to stain Mycobacteria /AFB which

do not stain well with Gram staining.
TWO METHODS

Method 1
• To stain strongly AFB : M. tuberculosis, M. bovis
• A 3% v/v HCl is used as decolorizer.

Method II
• To stain weakly AFB : M. leprae.
• A 1% v/v/ HCl is used as decolorizer.
 AFB-Smear Reporting

No. of AFB No. of AFB

ZN stain Auramine stain Report
(x1000) (x400)
0 0 No AFB seen

1-2/300 fields 1-2/70 fields +/- (request another specimen)

1-9/100 fields 2-18 / 50 fields +

1-9/10 fields 4-36 / 10 fields ++

1-9/field 4-36 / field +++

> 9 / field > 36 / field ++++

 ZIEHL-NEELSEN STAINING

PROCEDURE
1. Fixation of smear

2. Carbol fuchsin staining

° Cover the smear with carbol fuchsin stain.
° Heat the stain until vapours begin to rise.
° Do not overheat
° Do not let the stain to dry
° Wash off the stain with clean water.

3. Decolorization
° Cover the smear with 3% v/v/ acid-alcohol for 5 min OR
° Until the smear is sufficiently decolorized i.e. pale pink.
° Wash well with clean water
 ZIEHL-NEELSEN STAINING
4. Counterstaining
° Cover the smear with methylene blue stain for 1-2 min.
° Wash off the stain with clean water.
° Wipe the back of the slide clean and place in a draining rack
for the smear to air dry.
5. Microscopy
• Examine under 40X to see distribution of smear &
• Examine under oil immersion objective to look for AFB

RESULT
AFB Red coloured rods
Cells Bluish
Background Bluish
Fluorochrome Staining

• After staining with auramine & rhodamine stains :

• Examine by 40x objective

• AFB appear white yellow rods glowing against a dark

background when.
 ANTI-TB DRUGS

FIRST LINE DRUGS

• Isoniazid & bactericidal (active against intra

• Rifampicin and extracellular organisms)
3. Streptomycin bactericidal (active against
extracellular organisms only)
8. Pyrazinamide bactericidal (acts at acidic pH
within the cells only)
9. Ethambutol (bacteriostatic)

SECOND LINE DRUGS (added to combination if resistance or

toxicity contraindicate first-line ttt)
° Para-Aminosalicylic acid (PAS)
° Ethionamide
° CycloserineF-
° Fluoroquinolones
° Kanamycin, ect
 TREATMENT REGIMEN

• Drug combination with different modes of actions to delay

emergence of drug resistance.
• Isoniazid and Rifampicin + Pyrazinamide +Ethambutol
(for 6-9 months)
• Patients becomes non-infective after 2-3 weeks of
chemotherapy.
Long treatment period (slow response) is due to :
° Long doubling time of tubercle bacilli (12-24 h)
° Intra-phagocytic survival and multiplication.
° Metabolically inactive bacilli are not killed by drugs
° Caseous material interferes with drug action
CAUSES OF FAILURE OF CHEMOTHERAPY

1. Poor patient compliance

• Insufficient time and dosage

To improve patient’s compliance:

• A better approach is DOT (Directly Observed
Therapy Short Course) - patient takes drugs
under supervision

2. Resistant strains – MDR TB (Multi-drug

Resistant TB)
 PREVENTION OF TB
• Public health measures for early detection & treatment of TB
patients
• Eradication of TB in cattle’s and pasteurization of milk
• Chemoprophylaxis with INH to :
° Close contacts of TB patient
° Adults taking immuno-suppressive drugs
• BCG (Bacillus Calmette Guerin) vaccination
° BCG vaccine is a live-attenuated bovine strain
° Has been in use since 1923
GIVEN (by intra-dermal injection) TO :
° Tuberculin negative persons
° New born babies
NOT GIVEN TO:
° Persons with decrease cell-mediated immune mechanisms as in
AIDS
 Case study
• A nurse admitted to hospital complaining of a persistent fever,
general malaise and weight loss, over a period of 3 weeks. The
patient was place in isolation and a chest X-ray. Sputum culture and
microscopy were performed (see fig.)
 Questions
1. What is the name of the stain and culture medium used?
2. What is the identify of the organism?
3. What other methods exist for the diagnosis of this organism?
4. How would this infection managed?
 Case study
Ms F a 35-year-old child care worker from India, had been in a good health
until she began experiencing chronic cough and weight loss. Over two
months she developed gradually worsening cough productive of yellow
sputum. She also noted decreased appetite, a 8- kg weight loss, daily fever,
and night sweats. She did not improve despite being prescribed a routine
antibiotic for one week. A chest radiograph revealed infiltrates in both lung
apices. On further questioning she described that she worked briefly in an
AID ward in Madras, India. She also recalled her mother having a positive
tuberculin skin test about 20 years age, but her mother was never ill.
Ms F had a tuberculin skin test, but it showed no induration when examined
48 h later. Acid fast staining and microscopy of her sputum showed AFB.
She was placed on respiratory isolation at home and prescribed four anti-
tuberculous drugs. Within one week she felt much better, after two weeks
she was taken off respiratory isolation, and after one month was allowed to
return to work. She was cured after receiving six months of therapy, with
every dose administered under direct observation by a health department
nurse, as required by local public health authorities.
 The case raise several questions

1. How and when did Ms F most likely become infected with MTB?

3. Why did it take so long for Ms. F to develop active tuberculosis?

5. By what route did the tubercle bacilli most likely arrive at the
apex of her lung?

7. Which individuals are most likely to develop active tuberculosis

from contact with Ms F?

9. How would her illness have differed if she had AIDS?