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‫بسم ا الرحمن الرحيم‬

GENUS: MYCOBACTERIUM
Prof. Khalifa Sifaw Ghenghesh
 Obligate aerobe, Gram-positive rods
 Acid fast
 Complex cell wall lipids
– include mycolic acids
– protects vs. phagolysosomal components
 Peptidoglycan, glycolipids
– acid-fastness
 Two major groups:
– Slow growers:
– Rapid growers:
Mycrobacterium tuberculosis
 Non-motile, Non-sporing, non-capsulate
rods
 Grows on several enriched culture

media:
– Lowenstein-Jensen medium:
 Whole

egg, Glycerol, Asparagine, Mineral salts,
Malachite green

Mycobacterium bovis:
Lowenstein-Jensen Plate Culture Inoculated
with 15 Strains of Mycobacterium Species
CULTURE CHARACTERISTICS
 On primary isolation:

– visible growth after up to 8 weeks
 Colonies:
– Buff colour, dry bread crumb-like appearance
– Growth is eugonic (M. bovis = dysgonic)
 Growth temperature:
– 35-37oC
 Obligate aerobe
-------------------------------------------------------------------------- Heat-sensitive
 Susceptible to alcohol, glutaraldehyde and
formaldehyde.
Some differential characteristics of
tuberculle bacilli causing human disease
____________________________________________________
Species
Atmospheric
preference Nitratase TCH Pyrazinamide
--------------------------------------------------------------------------------------M. tuberculosis Aerobic
+
S
S
M. bovis

Microaerophilic

--

R

R

_______________________________________
TCH = thiophen-2-carboxylic acid hydrazide
S = sensitive, R = resistant
THE DISEASE
 Not highly contagious:
– transmission with prolonged contact
between susceptible and active case
– usually transmitted by airborne droplets,
must penetrate deep into respiratory tree
– infection can be via other routes:
ingestion

=> infection through cervical
or mesenteric LN
 Virulence
– Ability to Survive within Macrophages

 Primary Tuberculosis
 Post-Primary Tuberculosis
Stages of Primary Tuberculosis in Childhood
--------------------------------------------------------------------------------------Stage
Time (from onset)
Characteristics
--------------------------------------------------------------------------------------1.
3-8 weeks
Primary complex (PC)
develops and tuberculin
conversion occurs
2.
2-6 months
Progressive healing of PC,
possibility of pleural effusion
3
6-12 months
Possibility of miliary or
meningeal tuberculosis
4
1-3 years
Possibility of bone or joint
tuberculosis
5
3-5 years
Possibilty of genito-urinary or
chronic skin tuberculosis
Main differences between primary and post-primary
tuberculosis in the non-immunocompromised patients
--------------------------------------------------------------------------------Characteristics
Primary
Post-primary
--------------------------------------------------------------------------------Local lesion
Small
Large
Lymphatic involvement Yes
Minimal
Cavity formation
Rare
Frequent
Tuberculin reactivity
Negative (initially) Positive
Infectivity
Uncommon
Usual
(pulmonary cases)
Site
Any part of lung Apical region
Local spread
Uncommon
Frequent
---------------------------------------------------------------------------------------
TUBERCULIN TEST
 Tuberculin: a heat-concentrated filtrate of a
broth in which tubercle bacilli had been grown.
 Injection of tuberculin into the skin >>
– Large, indurated reactions >>Post-Primary
Tuberculosis.
– No induration >> Protective immunity

 Purified Protein Derivatives (PPD):
– Mantoux Method (Intracutaneous)
– Heaf Method (Spring-loaded gun)
– Tine Tests (Disposable single tests)
Mycobacteria-Positive PPD
LABORATOY DIAGNOSIS
1. Specimen:
– Pulmonary tuberculosis: > Sputum, Bronchial
washings, Laryngeal swabs, and Early-

morning gastric aspirates.
– Homogenized tissue biopsies.
– Examine after centrifugation: Deposits of
CSF, Pleural fluid, Urine and other fluids
2. Microscopy:
– Ziehl-Neelsen Stain
– Fluorescent dyes

3. Culture:
– Decontamination:
– Lowenstein Jensen medium

4. Nucleic Acid Methods:
Mycobacterium tuberculosis sputum
smear (Ziehl-Neelsen stain)
Mycobacterium tuberculosis in a
sputum smear (Ziehl-Neelsen stain)
Mycobacteria - Auramine Stained and Viewed with
Fluorescence Microscopy.
Acid Fast Bacilli Appear as Glowing Yellow Rods.
Histopathology of Tuberculosis, Endometrium.
Ziehl-Neelsen Stain.
An anteroposterior X-ray of a patient
diagnosed with advanced bilateral pulmonary
tuberculosis.
TREATMENT
Chemotherapy Recommended by International Union against
Tuberculosis and Lung Disease (examples)
--------------------------------------------------------------------------------------------------------Intial phase
Continuation
Regimen
(2 months)
phase (4 months)
Drug
Drug
--------------------------------------------------------------------------------------------------------HRZ
HR
Standard
HRZ
H3R3
Intermittent/ when
HRZ
H2R2
supervision is indicated
HRZE
HR
When there is a high
HRZS
HR
incidence of initial drug
resistance
---------------------------------------------------------------------------------------------------------
EPIDEMIOLOGY
 Transmission:
– Open Pulmonary Tuberculosis
– Crowdness in homes and workplaces

 Tuberculosis and AIDS
 Tuberculosis in Developing Countries:

– Africa
CONTROL
 Early Detection and Treatment of Open

Cases
 Reducing Overcrowding
 Vaccination:
– Bacille Calmette-Guerin (BCG)
– Not effective as control measure
Mycobacterium leprae
 Leprosy (Hansen's disease)
– A chronic intracellular infectious disease
unique to man (with few exceptions).
Usually not fatal.
 Never been cultivated in vitro
 Armadillos:
– 1010 bacilli/gram of diseased tissue
Lectures 5-6-Mycobacterium tuberculosis, M. leprae, Actinomycetes
M. leprae can be grown in mouse foot pads
or the nine-banded armadillo (picture).
PATHOGENESIS
 Schwan cell >> Nerve damage >>

Anaesthesia and Muscle paralysis >>
Gradual destruction of extremities
> Nasal bones and eyes
Immune reactions >>
– Severe and permanent nerve damage
THE DISEASE
 Manifestations of the disease depend on the

resistance of the host.
1. Tuberculoid: host is highly resistant,
clinical abnormalities limited to a few
peripheral nerves and adjacent skin areas,
tuberculoid granuloma
2. Lepromatous: host lacks resistance, all
tissues affected, foam cell granuloma
3. Borderline:
The feet become subject to bone damage and
deformity through unnoticed wounds and infection.
Serious infections can lead to amputations.
Victims of leprosy often suffer amputations of
fingertips and toes due to unfelt trauma.
X-rays of different stages.
Leprosy affects facial nerves > loss of blinking
reflex of the eye > dryness, ulceration, and
blindness.
Lepromatous lesions on human back
LABORATORY DIAGNOSIS
 Histological Examination of Skin Biopsies
 Detection of Acid-Fast Bacilli:

– In Nasal Discharges
– Scrapings from Nasal Mucosa
– Slit-Skin Smears
Superficial incisions in skin >>

Bacillary Index (BI):
Bacillary Index (BI)
 1+
 2+
 3+
 4+
 5+
 6+

1-10 bacilli/100 fields
1-10 bacilli/10 fields
1-10 bacilli/
field
10-100 bacilli/ field
100-1000 bacilli/ field
>1000 bacilli/ field

 Morphological Index (MI):
TREATMENT
WHO Recommendations for Multidrug Therapy
----------------------------------------------------------------------------------------------Type of
Drug
Dose Frequency
Total
Leprosy
(mg)
Duration
-----------------------------------------------------------------------------------------------

Paucibacillary

Rifampicin
Dapsone

600
100

Monthly, Superv.
Daily, unsuperv.

6 months

Multibacillary

Rifampicin
Dapsone

600
100
300

Monthly, Superv.
Daily, Unsuperv.
Mothly, Superv.

>2 years

+50

Daily, Unsuperv.

Clofazimine

{

-----------------------------------------------------------------------------------------------
EPIDEMIOLOGY
 Transmission:
– Nasal secretions of patients with lepromatous leprosy.
 Skin Test: Limited diagnostic value
– Lepromins > boiled-bacilli rich lepromatous lesions
– Leprosins > ultrasonicates of tissue-free bacilli from
lesions.

 Two Types of Reaction:
– Fernandez Reaction: sensitized individuals > 48h
(leprosin)
– Mitsuda Reaction: granulomatous swelling > ~3 weeks
(lepromin)
‫بسم ا الرحمن الرحيم‬

‫‪ACTINOMYCETES‬‬
 Gram +ve, Filaments that Break Up

Into Bacillary and Coccoid Forms.
 Non-Motile, Non-Sporing, Non-

Capsulated.
 Free Living >> Soil
1. ACTINOMYCES SPECIES
 A. israelii
– Actinomycosis >>
Chronic Granulomatous Infection.
– Formation of Sulphur granules:
 3 Forms:
i. Cervicofacial
ii. Thoracic
iii. Abdominal
 Predisposing Factors:
Trauma, Poor Oral Hygiene.
A. israelii
Gram stain showing diphtheroidal rods and
short branching filaments.
Actinomycosis-organisms aspirated from the lung. Long,
tortuous and branching organisms can easily be visualized
using silver stain. Sulfur granules not seen in aspirations.
LABORATORY DIAGNOSIS
i. Direct Examination:
Sputum, Pus, etc.. >> Examined
for Granules
ii. Culture: Brain Heart Infusion Agar

TREATMENT
– Penicillin >> Several Weeks
Sulfur granule from human actinomycosis tissue
section (hematoxylin and eosin stain).
2. NOCARDIA SPECIES
 N. asteroides
– Nocardiosis.
 Aerobic.
 Disease Begins as Pulmonary Infection >
 > 50% of Patients are Immunocompromised.
 Fatality Rate >>
LABORATORY DIAGNOSIS
i. Direct Examination:
 Sputum, Skin Lesions, Tissue Biopsies or Surgical Material

>> Microscopically.
 Observe: G+ve, Multiple Branched and Beaded Filaments.

> Partially Acid-Fast.

 ii. Culture:
 iii. Identification: Biochemically.

TREATMENT
 Sulphonamides, NA, TMP-SMX.
Nocardia asteroides
Silver stain showing the twisted masses of
long filamentous organisms
Nocardia asteroides
Acid fast stain shows the pale red staining
organisms in an area of necrosis
Differences between the genera
Actinomyces and Nocardia
-----------------------------------------------------------------Actinomyces species

Nocardia species

-----------------------------------------------------------------Facultative anaerobes
Grow at 35-37oC
Oral commensals
Non-acid-fast mycelia
Endogenous cause of
Disease

Strict aerobes
Wide temp range of growth
Environmental saprophytes
Usually weakly acid-fast
Exogenous cause of disease

------------------------------------------------------------------

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Lectures 5-6-Mycobacterium tuberculosis, M. leprae, Actinomycetes

  • 1. ‫بسم ا الرحمن الرحيم‬ GENUS: MYCOBACTERIUM Prof. Khalifa Sifaw Ghenghesh
  • 2.  Obligate aerobe, Gram-positive rods  Acid fast  Complex cell wall lipids – include mycolic acids – protects vs. phagolysosomal components  Peptidoglycan, glycolipids – acid-fastness  Two major groups: – Slow growers: – Rapid growers:
  • 3. Mycrobacterium tuberculosis  Non-motile, Non-sporing, non-capsulate rods  Grows on several enriched culture media: – Lowenstein-Jensen medium:  Whole egg, Glycerol, Asparagine, Mineral salts, Malachite green Mycobacterium bovis:
  • 4. Lowenstein-Jensen Plate Culture Inoculated with 15 Strains of Mycobacterium Species
  • 5. CULTURE CHARACTERISTICS  On primary isolation: – visible growth after up to 8 weeks  Colonies: – Buff colour, dry bread crumb-like appearance – Growth is eugonic (M. bovis = dysgonic)  Growth temperature: – 35-37oC  Obligate aerobe -------------------------------------------------------------------------- Heat-sensitive  Susceptible to alcohol, glutaraldehyde and formaldehyde.
  • 6. Some differential characteristics of tuberculle bacilli causing human disease ____________________________________________________ Species Atmospheric preference Nitratase TCH Pyrazinamide --------------------------------------------------------------------------------------M. tuberculosis Aerobic + S S M. bovis Microaerophilic -- R R _______________________________________ TCH = thiophen-2-carboxylic acid hydrazide S = sensitive, R = resistant
  • 7. THE DISEASE  Not highly contagious: – transmission with prolonged contact between susceptible and active case – usually transmitted by airborne droplets, must penetrate deep into respiratory tree – infection can be via other routes: ingestion => infection through cervical or mesenteric LN
  • 8.  Virulence – Ability to Survive within Macrophages  Primary Tuberculosis  Post-Primary Tuberculosis
  • 9. Stages of Primary Tuberculosis in Childhood --------------------------------------------------------------------------------------Stage Time (from onset) Characteristics --------------------------------------------------------------------------------------1. 3-8 weeks Primary complex (PC) develops and tuberculin conversion occurs 2. 2-6 months Progressive healing of PC, possibility of pleural effusion 3 6-12 months Possibility of miliary or meningeal tuberculosis 4 1-3 years Possibility of bone or joint tuberculosis 5 3-5 years Possibilty of genito-urinary or chronic skin tuberculosis
  • 10. Main differences between primary and post-primary tuberculosis in the non-immunocompromised patients --------------------------------------------------------------------------------Characteristics Primary Post-primary --------------------------------------------------------------------------------Local lesion Small Large Lymphatic involvement Yes Minimal Cavity formation Rare Frequent Tuberculin reactivity Negative (initially) Positive Infectivity Uncommon Usual (pulmonary cases) Site Any part of lung Apical region Local spread Uncommon Frequent ---------------------------------------------------------------------------------------
  • 11. TUBERCULIN TEST  Tuberculin: a heat-concentrated filtrate of a broth in which tubercle bacilli had been grown.  Injection of tuberculin into the skin >> – Large, indurated reactions >>Post-Primary Tuberculosis. – No induration >> Protective immunity  Purified Protein Derivatives (PPD): – Mantoux Method (Intracutaneous) – Heaf Method (Spring-loaded gun) – Tine Tests (Disposable single tests)
  • 13. LABORATOY DIAGNOSIS 1. Specimen: – Pulmonary tuberculosis: > Sputum, Bronchial washings, Laryngeal swabs, and Early- morning gastric aspirates. – Homogenized tissue biopsies. – Examine after centrifugation: Deposits of CSF, Pleural fluid, Urine and other fluids
  • 14. 2. Microscopy: – Ziehl-Neelsen Stain – Fluorescent dyes 3. Culture: – Decontamination: – Lowenstein Jensen medium 4. Nucleic Acid Methods:
  • 16. Mycobacterium tuberculosis in a sputum smear (Ziehl-Neelsen stain)
  • 17. Mycobacteria - Auramine Stained and Viewed with Fluorescence Microscopy. Acid Fast Bacilli Appear as Glowing Yellow Rods.
  • 18. Histopathology of Tuberculosis, Endometrium. Ziehl-Neelsen Stain.
  • 19. An anteroposterior X-ray of a patient diagnosed with advanced bilateral pulmonary tuberculosis.
  • 20. TREATMENT Chemotherapy Recommended by International Union against Tuberculosis and Lung Disease (examples) --------------------------------------------------------------------------------------------------------Intial phase Continuation Regimen (2 months) phase (4 months) Drug Drug --------------------------------------------------------------------------------------------------------HRZ HR Standard HRZ H3R3 Intermittent/ when HRZ H2R2 supervision is indicated HRZE HR When there is a high HRZS HR incidence of initial drug resistance ---------------------------------------------------------------------------------------------------------
  • 21. EPIDEMIOLOGY  Transmission: – Open Pulmonary Tuberculosis – Crowdness in homes and workplaces  Tuberculosis and AIDS  Tuberculosis in Developing Countries: – Africa
  • 22. CONTROL  Early Detection and Treatment of Open Cases  Reducing Overcrowding  Vaccination: – Bacille Calmette-Guerin (BCG) – Not effective as control measure
  • 23. Mycobacterium leprae  Leprosy (Hansen's disease) – A chronic intracellular infectious disease unique to man (with few exceptions). Usually not fatal.  Never been cultivated in vitro  Armadillos: – 1010 bacilli/gram of diseased tissue
  • 25. M. leprae can be grown in mouse foot pads or the nine-banded armadillo (picture).
  • 26. PATHOGENESIS  Schwan cell >> Nerve damage >> Anaesthesia and Muscle paralysis >> Gradual destruction of extremities > Nasal bones and eyes Immune reactions >> – Severe and permanent nerve damage
  • 27. THE DISEASE  Manifestations of the disease depend on the resistance of the host. 1. Tuberculoid: host is highly resistant, clinical abnormalities limited to a few peripheral nerves and adjacent skin areas, tuberculoid granuloma 2. Lepromatous: host lacks resistance, all tissues affected, foam cell granuloma 3. Borderline:
  • 28. The feet become subject to bone damage and deformity through unnoticed wounds and infection. Serious infections can lead to amputations.
  • 29. Victims of leprosy often suffer amputations of fingertips and toes due to unfelt trauma. X-rays of different stages.
  • 30. Leprosy affects facial nerves > loss of blinking reflex of the eye > dryness, ulceration, and blindness.
  • 32. LABORATORY DIAGNOSIS  Histological Examination of Skin Biopsies  Detection of Acid-Fast Bacilli: – In Nasal Discharges – Scrapings from Nasal Mucosa – Slit-Skin Smears Superficial incisions in skin >> Bacillary Index (BI):
  • 33. Bacillary Index (BI)  1+  2+  3+  4+  5+  6+ 1-10 bacilli/100 fields 1-10 bacilli/10 fields 1-10 bacilli/ field 10-100 bacilli/ field 100-1000 bacilli/ field >1000 bacilli/ field  Morphological Index (MI):
  • 34. TREATMENT WHO Recommendations for Multidrug Therapy ----------------------------------------------------------------------------------------------Type of Drug Dose Frequency Total Leprosy (mg) Duration ----------------------------------------------------------------------------------------------- Paucibacillary Rifampicin Dapsone 600 100 Monthly, Superv. Daily, unsuperv. 6 months Multibacillary Rifampicin Dapsone 600 100 300 Monthly, Superv. Daily, Unsuperv. Mothly, Superv. >2 years +50 Daily, Unsuperv. Clofazimine { -----------------------------------------------------------------------------------------------
  • 35. EPIDEMIOLOGY  Transmission: – Nasal secretions of patients with lepromatous leprosy.  Skin Test: Limited diagnostic value – Lepromins > boiled-bacilli rich lepromatous lesions – Leprosins > ultrasonicates of tissue-free bacilli from lesions.  Two Types of Reaction: – Fernandez Reaction: sensitized individuals > 48h (leprosin) – Mitsuda Reaction: granulomatous swelling > ~3 weeks (lepromin)
  • 36. ‫بسم ا الرحمن الرحيم‬ ‫‪ACTINOMYCETES‬‬
  • 37.  Gram +ve, Filaments that Break Up Into Bacillary and Coccoid Forms.  Non-Motile, Non-Sporing, Non- Capsulated.  Free Living >> Soil
  • 38. 1. ACTINOMYCES SPECIES  A. israelii – Actinomycosis >> Chronic Granulomatous Infection. – Formation of Sulphur granules:  3 Forms: i. Cervicofacial ii. Thoracic iii. Abdominal  Predisposing Factors: Trauma, Poor Oral Hygiene.
  • 39. A. israelii Gram stain showing diphtheroidal rods and short branching filaments.
  • 40. Actinomycosis-organisms aspirated from the lung. Long, tortuous and branching organisms can easily be visualized using silver stain. Sulfur granules not seen in aspirations.
  • 41. LABORATORY DIAGNOSIS i. Direct Examination: Sputum, Pus, etc.. >> Examined for Granules ii. Culture: Brain Heart Infusion Agar TREATMENT – Penicillin >> Several Weeks
  • 42. Sulfur granule from human actinomycosis tissue section (hematoxylin and eosin stain).
  • 43. 2. NOCARDIA SPECIES  N. asteroides – Nocardiosis.  Aerobic.  Disease Begins as Pulmonary Infection >  > 50% of Patients are Immunocompromised.  Fatality Rate >>
  • 44. LABORATORY DIAGNOSIS i. Direct Examination:  Sputum, Skin Lesions, Tissue Biopsies or Surgical Material >> Microscopically.  Observe: G+ve, Multiple Branched and Beaded Filaments.  > Partially Acid-Fast.  ii. Culture:  iii. Identification: Biochemically. TREATMENT  Sulphonamides, NA, TMP-SMX.
  • 45. Nocardia asteroides Silver stain showing the twisted masses of long filamentous organisms
  • 46. Nocardia asteroides Acid fast stain shows the pale red staining organisms in an area of necrosis
  • 47. Differences between the genera Actinomyces and Nocardia -----------------------------------------------------------------Actinomyces species Nocardia species -----------------------------------------------------------------Facultative anaerobes Grow at 35-37oC Oral commensals Non-acid-fast mycelia Endogenous cause of Disease Strict aerobes Wide temp range of growth Environmental saprophytes Usually weakly acid-fast Exogenous cause of disease ------------------------------------------------------------------