Mycobacterium

(Fungus Bacterium)
Classification: (Bergey s Manual of Systematic Bacteriology)
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Kingdom II: Bacteria Phylum VIII: Firmicutes Section XXIII: The Actinobacteria Class I: Actinobacteria (including: Actinomyces, Corynebacteria, Norcardia, Rhodococcus) Order V: Actinomycetals Suborder III: Corynebacterinaea Family IV: Mycobacteriaceae Genus: Mycobacterium (only 1 genus, but >100 species!)

To include a species under this genus, the minimum standard is:
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Acid (alcohol) fastness :- Resists decolourization by acidified alcohol after stained with basic fuschin) Presence of mycolic acid (in cell wall) A G+C (Guanine + Cytosine) content of DNA of 61-71 mol %

Genus Mycobacterium
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At least 100 species to date! Causative agents for i. Tuberculosis ii. Leprosy iii. Similar diseases in animals iv. Some saprophytes opportunists

Tuberculosis
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Disease of great antiquity Afflicted Neolithic man (circa 10,000B.C) Described in Vedas as Raja-Yakshma (~King of diseases) Well described by Hippocrates (400 B.C) Caused more suffering & death than any other infection Was referred to as the White plague

History
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Mycobacterium among first bacteria discovered 1874: Armauer Hansen Bacillus Leprae 1882: Robert Koch Bacillus Tuberculosis 1884: Robert Koch Koch s Postulates Subsequently bacillus Mycobacterium (mould-like pellicle)

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1900 s: Other spp. Discovered (opportunistic, environmental, non-tuberculous)

Mycobacterium: Acid Fast Bacilli Standard staining methods
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Ziehl-Neelsen (hot staining) Kinyoun (cold staining) Auramine o fluorescence

Ziehl Neelsen Stain
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Basic fuchsin + phenol (enhance fuchsin penetration into lipids) Wash Acid alcohol (decolorizer) Wash Methylene Blue (Counter stain)

Other Acid Fast Organisms
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Norcardia Actinomyces Corynebacterium Rhodococcus

Culture
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M. Leprae HAS NOT BEEN GROWN ON CULTURE! Others: - Lowenstein Jensen (L J) medium - Middlebrook 7H9 (liquid) - Middlebrook 7H10 (Agar based) - Middlebrook 7H11 (Agar based too!) Clinical specimens : 7 days 6 weeks (Sometimes, even up to 12 weeks or more!) As a whole, mycobacterium are VERY slow growing

Mycobacterium Acid Fast Bacilli
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Non-motile Non-spore forming Weakly Gram (+) Aerobic or microaerophilic (M. Tuberculosis is strictly aerobic) Straight or slightly curved rod-shaped Some coccobacillary - Filamentous - Branched forms Some spp produced pigments (yellow orange) - In the dark (scotochromogens)

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- After exposure to light (photochromogens) - No-pigmentation (Non-chromogens) Some slow growers (>7/7 to form visible colonies) Some rapid growers (<7/7) 1959 Runyon divided mycobacterium into 4 groups i. Group I = Photochromogens ii. Group II = Scotochromogens iii. Group III = Non-Chromogens iv. Group IV = Rapid growers Groups I-III are slow growers

Mycobacterium:
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Can be isolated from a variety of sources - Clinical specimens - Environment (water, soil, dust) Can survive for long periods of time As pathogens i. Obligate pathogens a. M. tuberculosis b. M. bovis c. M. africanum d. M. ascaticum e. M. farcinogens f. M. haemophilum ii. Facultative pathogens a. M. avium b. M. chelonae c. M. fortuitum d. M. kansasii e. M. ulcerans f. M. terrae g. M. genavense

g. h. i. j. k. l. m.

M. leprae M. malmoense M. microti M. paratuberculosis M. shimodei M. Simiae M. Szulgai

h. i. j. k. l. m.

M. intracellulare M. marinum M. senegalense M. scrofulaceum M. xenopi M. malmoense

TUBERCULOSIS
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Chronic granulomatous disease Affects humans and many mammals At one time single most important infectious disease of humans ( 1/7th of all deaths worldwide) Presently: - 1/3 world population infected - Cause about 3 million deaths per year (>5% of all deaths) - Is becoming a reemerging disease because of HIV Caused by 4 very closely related spp:

i. ii. iii.

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M. tuberculosis (Humans) M. bovis (Cattle) M. africanum (intermediate of above) a. Type I: In West Africa (~M. bovis) b. Type II: In East Africa (~M. Tuberculosis) iv. M. microti (Vole) Not in man M. canetti (Very rare M. TB c smooth) M. bovis variant in good few TB in vets (M. caprae)

By the way, this is a vole:

Mycobacterium Tuberculosis
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Acid fast bacteria Obligate aerobe Non-motile Non-sporing Unencapsulated Straight / Slightly curved rod 3 x 0.3 µm in size Grow in several enriched media LJ most widely used (Whole egg, glycerol, asparagine, salt) + Malachite green (inhibit contaminants kills other microorganisms) M. TB Obligate aerobe (on surface) Heaped up luxuriant eugonic + ++ S R M. bovis Micro-aerophilic (few mm below surface) Small flat dysgonic +/R S M. africanum Micro-aerophilic Intermediate Variable +/S S

Atmospheric preference Growth in LJ Reduce nitrate Nitrite Niacin production Sensitivity to pyrazinamide Sensitivity to thiopen-2

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TB

Primary

Post primary (or secondary

Pri ary TB
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Bacilli Alveolar microorganisms in lungs multiply Initial lesion



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P rt of entry Respiratory tract Mode Inhalation of bacilli Site Lungs (usually

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ulosis

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Initial lesion (Ghon Focus)

Hilar lymph nodes enlarges

Other parts of lungs

Hematogenous Spread

Lymphatic Spread

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Usually limit primary infection

Granuloma with central caseation Quiescent Fibroblasts Scar + Calcification

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1-2 weeks

T-cell clones (Antigen specific)

Cytokines

Macrophage activation

Granuloma Formation

Central Caseation (Cheese like in appearance & consistency)

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If part of entry mouth (milk, food) = Primary comple y Tonsil y Cervical lymph node enlarged (Scrofula) y Intestine (ileocaecal region + mesenteric lymph node) If part of entry skin y Skin + regional lymph nodes = Primary comple (Prospecter s
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NOT ALL hosts can completely destroy mycobacterium Dormant (How??) = immunocompromised Reactivation (?? Reinfection) Post-primary TB STAGES OF PRIMARY TB IN CHILDHOOD Time from onset 3-6 weeks 2-6 months 6-12 months 1-3 years 3-5 years Characteristics y Primary complex develops y TB Conversion + y Progressive healing of primary complex y Possibility of pleural effusion y Possibility of miliary or meningeal TB y Possibility of bone or joint TB y Possibility of gastro-urinary or chronic TB

Just for fun Balita = Bawah Lima Tahun

POST PRIMARY TB
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Most of those exposed to M. TB = Primary complex heals completely y Only evidence (+) = Tuberculin test (+) In some, after months/years/decades = Reactivation or ?? Exogenous infection Post-primary TB - Larger local lesions - Minimal lymphatic involvement - > open cases infective - Especially apical region of lungs Reactivation - Spontaneous - Decrease immune responsiveness Process of granuloma formation = Same except more extensive with large areas of caseation (tuberculomata)  Proteases & Cytokines (e.g. TNF) Process = Erode wall of bronchus Liquefied content Expectorated Aerobic (open cases) Bacilli growth In immunocompromised: - Cavitation rare! - Lymphatic & hematogenous spread common Cryptic disseminated TB

Diagnosis
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Clinical X-ray Tuberculin test - Old tuberculin

PPD activities 1 100 100 iu } 1 1000 10 iu } 0 1 ml 1 10000 1 iu } Standard : 10 iu iu = international unit (+) Induration <5 mm 5-10 mm >10 mm y y y y Interpretation ndemic areas on-endemic areas Children Immunocompromised Lab specimens = Sputum, bronchial washings, biopsies, gastric aspirates, CSF Pleural fluid, etc Microscopy Culture
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Sputum

Decontaminate with NaOh (4%)

Deposit

Inoculate onto culture media (LJ or middlebrook's)

If CSF or Pleural Fluid or Biopsy, no need to decontaminate or centrifuge Directly deposit and inoculate
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Purified protein derivative (PP ) Mantoux test Intracutaneously Heaf test Gun with 6 prongs Tine test Dried PPD onto prongs for 1 test (disposable)

Centrifuge

After 7 days, read (growth)

DRUG SUSCEPTIBILITY TESTING
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In L.J, containing doubling dilutions of drugs

TREATMENT Drugs = 3 groups 1. Sterilizing - Rifampin - Pyrazinamide 2. Bacteriostatic - Ethionamide - Prothionamide - Thiacetazone - P-aminosalicylic acid - Cycloserine 3. Bacteriocidal - Isoniazid - Streptomycin - Ethambutol Also: Multidrug treatment (MDT) Other ways to Diagnose TB
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-PGL (glycolipid) PCR BCG (Bacillus Calmette-Guerin) Protects children for severe TB Offers cross protection against Leprosy

Leprosy
(Morbus Hansen [MH]) (Hansen s disease) Definition: A chronic mycobacterial disease primarily affecting the peripheral nervous system and secondarily involving skin and other tissues.

History
600 B.C written records in India 150 B.C written records in China 2nd century BC Egyptian mummies From Egypt -> Europe (150 AD: Greece) Then -> Americas & other parts of the world 1983: WHO -> Estimated total in world 10.5 million

1996 (Multiple drug treatment) -> 1.3 million Malaysia Common Orang Asli & Foreign workers

Etiology
Mycobacterium Leprae [unculturable in lab] (Armauer Hansen 1874) Grown in: mouse footpad 9 banded armadillos (discovered because of Armauer Hansen s wife)

Man only significant reservoir Transmission: via nasal secretions by droplets Skin

Pathogenesis
Principal target cell = Schwann cells Nerve damage Anaesthesia Muscle paralysis Skin -> usually first sign/symptom Non-specific lesions with pigmentary changes (hypopigmented) anaesthesia / hypoaesthesia (Whether the patient still feels pain at the area. Use a needle to prick) Often heals spontaneuously Clinical manifestations depend on immune status of patient Range TUBERCULOID -> LEPROMATOUS TUBERCULOID Hyperactive immune response Few localized skin lesions with few bacilli LEPROMATOUS Anergy Numerous skin lesions often confluent

(paucibacillary) Intense granulomatous response -> Damage major nerves (hardening) Bacilli ++++ (multicacillary)

Immune response greatly weakened (anergy) Ear lones bacilli ++++

Those with the better immune system will suffer from the tuberculoid form. Those with the lepromatous form will have a negative tuberculin test. The safest area to wear earings/studs is at the ear lobes. Anywhere else, then it will easily affect the ear cartilage. It could lead to cauliflower ear. (Pak Nasa s story) More WHO criteria: Borderline tuberculoid (BT) Mid-Borderline (BB) Borderline Lepromatous (BL)

Other signs & Symptoms
Eye Uveitis

Corneal infection secondary to eyelid paralysis The 2 symptoms above might lead the person to suffer from blindness Bone resorbtion Collapse of nasal bone Shortening of fingers & toes (autoamputation) Neuritis Orchitis Immune-Complex Nephritis Facial skin may become crumpled and look like a lion (Facies leonina)

DIAGNOSIS
History & physical examination Slit-skin smear and punch biopsy (AFB) Ear lobe Nasal secretion Nasal mucosa Histology granuloma + caseation Skin test Lepromin test (only guide) Fernandez (early) Rx 1-2/7 Mutsida (late) Rx 7/7 (Usually negative in Lepromatous Leprosy) Most cases of Lepromatous Leprosy cases in Malaysia are among the Chinese

Split-skin smear - Acid fast staining
Record # of bacteria/high power 1+ : 1-10 per 100 fields 2+ : 1-10 per 10 fields 3+ : 1-10 per field 4+ : 10-100 per field 5+ : 100-1000 per field 6+ : >1000 per field = Bacillary Index (BI)

Acid Fast Bacilli: if viable -> stains strongly and evenly Dead when stains unevenly & weakly Morphological Index (MI): Percentage (%) of regularly stained (viable) Increase MI Active disease MI decreases with chemotherapy Other Diagnosis methods: -PGL Polymerase Chain Reaction (PCR) Treatment: Dapsone (diaminophenyl sulphone DDS) Before 1982 Standard monotherapy for all forms DDS Resistance -> MDT Add Rifampin Clofazimine (side effect: Skin discolouration) Other drugs Prothionamide Ofloxacin Minocycline WHO Paucibacillary Rifampin Dapsone (Give these for 6 months) Multibacillary Rifampin Dapsone Clofazimine (Given for 2 years or more) Reactions: Lepra/Jopling s Type I: Delayed Type Hypersensitivity Vasculitis (Type II HS) Lepra/Jopling s Type II Erythema Nodosum Leprosum Infections by environmental (Opportunistic) mycobacteria (Pak Nasa said this part is not important)

Runyon s classification Photochromogens (Group I) M. Kansasii M. Marinum M. Simiae b. Scotochromogens (Group II) M. Scrofulaceum M. gordonae M. szulgai c. Non-Chromogens (Group III) M. avium M. intercellulare M. malmoense M. xenopi M. ulcerans M. terrae d. Rapid Growers (Group IV) M. chelonae Principal types of opportunistic mycobacterial infections in man
a.

Lymphadenopathy M. Avium complex M. Scrofulaceum Skin lesions Post-trauma abscesses M. Chelonae M. Fortuitum M. Terrae b. Swimming Pool Granuloma M. Marinum c. Buruli ulcer M. Ulcerans Pulmonary Disease
a.

M. Avium complex M. Kansasii M. Xenopi M. Malmoeure Disseminated Disease AIDS related M. Avium complex M. Genevense b. Non-AIDS related M. Avium complex M. Chelonae
a.