MYCOBACTERIUM TRANS NO.

Summary: Manual , Lecture (Dr. Santos)

Date of lecture: September 22, 2012
Transcriptionist: Xanne

MYCOBACTERIUM
Group IV M. fortuitum Rapid (-)
- rod-shaped aerobic bacilli M. chelonei (4)
- neither Gram-positive or negative M. smegmatis
- acid fast due to mycolic acid M. flavescens
- do not stain readily once stained resistant to
(1) Pigment formed in the presence of light only
decolorization with acid or alcohol ACID FAST
(2) Pigment formed in the presence or absence of light
- decolorizer is acid alcohol (95% ethyl alcohol + 3% HCl)
(3) Non-pigmented
- Cell wall: waxy, hydrophobic and high lipid contents
(4) Growth < 1 week
60% of dry weight
- Adjuvant properties responsible for development of
delayed type hypersensitivity The Global Emergency
- ALL mycobacterial pathogens are intracellular
8-12M new infections per year
pathogens
2-3M people die from TB per year
major pathogens:
Emergence of MDR M. Tuberculosis
o M. tuberculosis (Kochs bacillus)
th
Philippines is the 9 out of the 22 highly burdened
o M. leprae (Hansens bacillus) countries

th
o M. bovis Philippines is the 8 out of the 25 high MDRTB cases
Potentially pathogenic:
o M. avium complex
Mycobacterium tuberculosis
o M. kasasii
o M. fortuitum Morphology
MOTT (Mycobacterium Other Than Tuberculosis) - acid fast bacilli
- Anonymous, atypical, unclassified Mycobacteria, - non-motile, non-sporogenous, non-encapsulated
NMT (Non-tuberculose Mycobacteria) - arranged singly or in groups
- long, slender rods, beaded
Ranyoun Classification
- much granules in grams staining
Representative Growth Pigment Niacin
Species - easily destroyed by pasteurization
TB M. tuberculosis slow Pigmented (+) - Cord formation when obtained in liquid culture
Complex M. africanum medium
M. bovis - Requires glycerolated agar or potato medium
- Stained by acid-fast stains (Ziehl-Neelsen, Kinyoun) or
Group I M. asiaticum Slow Photo- (-) flourochrome stain
M. kasasii chromogens
- Culture: Non-pigmented, rough, irregular, wrinkled,
M. simiae (1)
M. marinum may show cauliflower-like appearance

Group II M. flavescens Slow Scoto- (-) Physiology

M. scrofulaceum chromogens - obligate aerobes
M. gordonae (2) - growth enhanced by O2 tension
M. szulgai - Lowenstein-Jensen or BACTEC
- Small, dry, scaly colonies with corrugated surfaces
Group III M. avium Slow Non- (-)
M.haemophilium chromogens - champion slow grower
M. ulcerans (3) o generation time: 13 15 hours
M. celatum o doubling time: 18 hours
M. gastri o 9 8 weeks before reported as negative
M. xenopi culture
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 - resistant to drying survive in the environment for
long periods of time
- one of the most resistant among non-sporeformers
- may result in familial infection
Constituents of the Cell Wall
A. Lipids
a. Mycolic acid
b. Waxes
c. Phosphatides
o mycolic acid + muramyl dipeptide (from
peptidoglycan) granuloma formation
o phospholipids induce caseation necrosis
B. Proteins
a. elicit the tuberculin reaction
b. elicit formation of antibodies
C. Polysaccharides
a. induce immediate type of hypersensitivity
b. serve as antigens in reaction with sera of
infected persons
Virulence Factors
- NO toxins
A. Cord Factor
o trehalose-6,6-dimycolate
o responsible for serpentine cord formation
(bacilli arranged in parallel chains)
o inhibits migration of PMNs
o elicits granuloma formation
o serve as immunologic adjuvant
B. Sulfatides
o synergistically potentiate toxicity of cord
factor
o promote survival of bacilli inside macrophages
C. Antibacterial resistance
Pathogenicity and Pathogenesis
- among the top 10 disease killers in the Philippines
- predisposing factors:
o chronic fatigue
o sedentary life
o malnutrition
o poor housing conditions
o occupational
Modes of Transmission
- inhalation of droplet nuclei (coughing or sneezing)
o 1 org enough to establish infection
- ingestion of unpasteurized milk or milk products
- kissing
- fomites
- skin contact
Pathogenesis
Inhalation of droplet nuclei -> deposit in alveolar space
of lungs -> engulfed by macrophages
Portion: resist intracellular destruction -> persist ->
multiply and kill the macrophages stimulate and
inflammatory focus -> mature into a granulomatous
lesion (tubercle) -> CASEOUS NECROSIS -> erosion of
tubercle into an adjacent airway -> cavitation ->
release of massive numbers of bacilli into the sputum
Resistant host: tubercle -> calcified
Early infection:
o Spread distally
o Indirectly thru lymphatics -> hilar or
mediastinal LN
o Directly into the circulation by erosion into
pulmonary vessel
*Infection with the tubercle bacillus usually involves
the lungs, but any area of the body can be involved.
Principal Types of Lesions
A. Exudative Type
o consists of inflammatory reaction, PMN
leukocytes & later monocytes around the
tubercle bacilli
o particularly seen in lung tissue
o may be absorbed or lead to massive necrosis
of tissue or may develop into productive type
o Tuberculin test (+)
B. Productive Type
o chronic granuloma
o consists of 3 zones
a. central area of multinucleated giant cells
containing tubercle bacilli
b. midzone of epithelioid cells
c. peripheral zone of fibroblasts,
lymphocytes & monocytes
o peripheral zone develops fibrous tissue
o central area is site of caseation necrosis
tubercle break into bronchus & empty
contents cavitation
o healing by fibrosis or cavitation
o the production, development, and healing or
progression are determined by:
number of bacilli in the inoculum
resistance & hypersensitivity of the host
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 - lymph node undergoes massive caseation calcifies
Extrapulmonary Tuberculosis - (+) Tuberculin test
- 42% - lymphatics - most often involve the base of the lung
- 21% - pleural
- 11% - bone / joint Reactivation
- 10% - others - tubercle bacilli survive the primary lesion
- 7% - genitourinary - characterized by chronic tissue lesions, tubercle
- 5% - meningeal formation, caseation & fibrosis
- 4% - peritoneal - regional lymph nodes are only slightly involved without
caseation
Once infected by the tubercle bacillus, one of the two things - located at the apex of the lung
will occur:
1. Latently Infection Immunity
o the individuals immune system contains the 1. Humoral Immunity
spread of infection o antibodies non-protective
2. Active Tuberculosis 2. Cellular Immunity
o the individuals immune system is unable to o activated CD4 & TH1 release large amounts
contain the infection of gamma interferon activate macrophage
destroy intracellular bacilli
o growth and spread of tubercle bacilli is
Individuals or Groups at Risk of Tuberculosis
interfered with
1. Close contacts of persons or known suspected to have
3. BCG Vaccination
TB
o no absolute immunity, only relative immunity
2. People born outside of the US, from areas that have a
o there is less incidence or tuberculosis among
high incidence of TB
BCG vaccinated individuals
3. Residents & employees of high-risk congregate settings
4. Health care workers who serve high-risk clients
Tuberculin Test
5. Some medically underserved, low income populations, 1. Antigens
defined locally as having an increased prevalence of TB a. Old Tuberculin (OT)
6. Infants, children, & adolescents exposed to adults in b. Purified Protein Derivative (PPD)
high-risk categories 2. Older Antigens
7. Persons who inject illicit drugs & other locally a. Old Filtrate (BF)
identified high-risk substance abusers like crack b. Bacillary Emulsion (BE)
cocaine users c. Tuberculin Residuum (TR)
d. Synthetic Old Tuberculin Trichloroacetic
Individuals with Higher Probability of Progressing to Active
Precipitate (SOTT)
Disease
1. Persons with HIV infection 3. Methods
2. Persons who were recently infected with a. Mantoux intradermal
M. tuberculosis (within the past two years), particularly b. Tine disposable kit
infants and very young children c. Koch subcutaneous
3. Persons who have certain medical conditions known to d. Volmer patch
increase the risk of disease if infection occurs e. Von Pirquet cutaneous
4. Persons who inject illicit drugs and other locally f. Moro percutaneous
identified high-risk substance abusers like crack
cocaine users Reactions to Tuberculin
5. Persons with history of inadequately treated TB - skin test must be read in 24 72 hours
- (+) skin test induration of > 10 mm, erythema &
Primary Infection edema
st
- 1 contact with tubercle bacilli - (+) test persists for several days
- acute exudative lesion develops lymphatics & - (+) test does not indicate active disease or immunity to
regional lymph nodes often heals rapidly the disease
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 - within 4 6 weeks after infection (+) test cold method
- after BCG vaccination (+) test that lasts for 3 7 counterstain malachite green
years
- false negative results 2. Fluorochrome Stain
o general anergy
o Hodgkins disease Biochemical Tests
- Niacin test: (+)
o sarcoidosis
- Nitrate reduction: (+)
o AIDS
- Catalase test: ( - )
o immunosupression

PCR (Polymerase Chain Reaction)

- 55 90% sensitive
Clinical Findings
- afternoon rises in body temperature - 99% specific
- fatigue
Antibiotic Susceptibility Testing
- weakness
- very important due to increasing number of multi-drug
- loss of appetite
resistant strains
- weight loss
- chronic cough of more than 2 weeks with or without Other Tests
hemoptysis usually associated with far-advanced TB - gas chromatography
- mycodot rapid method
Diagnosis o 70% sensitivity
- specimens
o 96% specificity
o fresh sputum
- nucleic acid hybridization
o gastric washings
o excellent specificity & sensitivity
o urine
o very expensive
o CSF
o requires technical expertise
o blood
o biopsy material Treatment
st
- 1 Line Anti-TB Drugs
Specimen Selection & Collection o Isoniazid, Rifampicin, Ethambutol,
- volume
Pyrazinamide
- number of specimens
o used for 6 months to 1 year
o one specimen per day for 3 5 days
o DOTS (Direct Observation Therapy Short-
- timing
term)
o early morning specimens
useful to ensure patient compliance
Culture nd
- 2 Line Anti-TB Drugs
1. Lowenstein Jensen medium
o Cycloserine, Ethionamide, Paraaminosalicylic
o egg-based
acid, Vancomycin, Capreomycin,
2. Middlebrook medium
Fluoroquinolones, Kanamycin, Rifambutin
o agar-based or broth
o Streptomycin not used anymore because
o
*Incubate at 35 37 C in 5 10% CO2 for up to 8 weeks of CN VIII deafness

Staining
1. Acid Fast Stain Drug-Resistant Tuberculosis
Two Methods - in the 1900s, multi-drug resistant tuberculosis (MDR-
a. Ziehl Neelsen method TB), defined as resistant to Isoniazid & Rifampicin
hot method began to emerge both in the US & worldwide
conterstain methylene blue - today 400,000 cases of MDR-TB globally
b. Kinyoun method - outbreak of extensively drug-resistant tuberculosis
(XDR-TB) MDR-TB with additional resistance of
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 Fluroquinolones & at least one of the following
injectibles: Capreomycin, Amikacin, or Kanamycin in
South Africa & in over 40 countries
- estimated nearly 30,000 XDR-TB cases globally
Neurologic Disturbances
Why is there so much drug-resistant tuberculosis?
Drug resistance is created when patients with
tuberculosis are not treated adequately either because:
a. The patient does not take the drug treatment
regimen as prescribed (nonadherent)
b. The treating physician or program does not treat
the patient with the appropriate drug regimen
- discrete erythematous, infiltrated nodules
1 5 cm in diameter
- diffuse skin infiltration
- nerve infiltration and thickening anesthesia,
neuritis, paresthesia, trophic ulcers, bone
- resorption and shortening of digits
- disfigurement (leonine facies) due to skin infiltration
and nerve involvement
- saddle nose due to bone resorption

Treatment Regimen Two Major Types

- patients with drug susceptible TB four-drug regimen Tuberculoid Lepromatous
st
that is administered for 6 months 1 line drugs Features
Leprosy Leprosy
Isoniazid, Rifampin, Ethambutol & Pyrazinamide
st erythematous,
- after 1 2 months D/C Pyrazinamide & Ethambutol macular,
Lesion infiltrated nodules,
continue Isoniazid & Rifampicin for another 4 hyperpigmentated
diffuse
months to complete the 6-month regimen
- medications can be administered daily, twice a week, Amount of
paucibacillary multibacillary
or three times a week, with similar results bacteria
- the expected outcomes with this treatment regimen
Patchy
are excellent with few treatment failures & relapse localized diffuse
anesthesia
rate of only 2 3%
Disfigurement absent present

Lepromin test positive negative

Mycobacterium leprae

Appearance of Disease
- acid-fast bacilli
- singly, parallel bundles, or globular masses Lepromatous Tuberculoid
- regularly found in scrapings from skin or mucous Deficient CMI Intact CMI
membranes (nasal septum) of leper Progressive disease Non-progressive disease
Nodular skin lesions Macular skin lesions
LEPROSY (Hansens disease)
Abundant bacilli in Few bacilli in lesions
- incubation period is short (few days) up to 40 years lesions
- onset is insidious Symmetric nerve Severe asymmetric nerve
- involves the cooler tissue of the body: involvement involvement
o skin Slow onset Sudden onset
o superficial nerves IL-4 and IL-10 IL2 IFN gamma and IL12
o nose Skin is infiltrated with Skin infiltrated with Th1
o pharynx suppressor T cells cells
Ab levels are high
o larynx
Malignant Benign
o eyes
Continuous
o testicles bacteremia
- mode of transmission is through prolonged contact Negative lepromin, Positive lepromin skin test
and exposure extract of
lepromatous tissue,
Skin Lesions skin test
- pale, anesthetic macular lesions (1 10 cm in
diameter)

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Diagnosis / Clinical Findings
- acid fast staining of skin lesions or nasal scrapings
- Leromin test for tuberculoid leprosy
o Fernandez reaction early reaction
o Mitsuda reaction late reaction
- no serologic test is useful
- cannot be grown in any artificial laboratory medium

Treatment
- Dapsone (Diaminophenylsulfone)
o mainstay of therapy
- Dapsone + Rifampicin + Clofazimine
o drug combination is used in case of an
emerging drug resistance
- 2 years duration

Prevention
- isolation of patients
- for exposed children and household members
chemoprophylaxis Dapsone

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