5. Mycobacterium

8/7/2019 5. Mycobacterium

http://slidepdf.com/reader/full/5-mycobacterium 1/14

Mycobacterium

(Fungus Bacterium)

Classification: (Bergey s Manual of Systematic Bacteriology)

y Kingdom II: Bacteriay Ph ylum VIII: Firmicutesy Section XXIII: Th e Actinobacteriay Class I: Actinobacteria (including: Actinomyces, Corynebacteria, Norcardia, R h odococcus)y O rder V: Actinomycetalsy Suborder III: Corynebacterinaeay Family IV: Mycobacteriaceaey G enus: Mycobacterium (only 1 genus, but >100 species!)

To include a species under t h is genus, t h e minimum standard is:

y Acid (alco h ol) fastness :- Resists decolourization by acidified alco h ol after stained wit h basicfusc h in)

y P resence of mycolic acid (in cell wall)y A G+ C (G uanine + Cytosine) content of DNA of 61-71 mol %

G enus Mycobacterium

y At least 100 species to date!y Causative agents for

i. Tuberculosisii. Leprosyiii. Similar diseases in animalsiv. Some saprop h ytes opportunists

Tuberculosis

y Disease of great antiquityy Afflicted Neolit h ic man (circa 10,000B.C)y Described in Vedas as Raja-Yaks h ma (~King of diseases)y Well described by Hippocrates (400 B.C)y Caused more suffering & deat h th an any ot h er infectiony Was referred to as t h e Wh ite plague

H istory

y Mycobacterium among first bacteria discoveredy 1874: Armauer Hansen Bacillus Lepraey 1882: Robert Koc h Bacillus Tuberculosisy 1884: Robert Koc h Koc h sP ostulatesy Subsequently bacillus Mycobacterium (mould-like pellicle)



y 1900 s: O th er spp. Discovered (opportunistic, environmental, non-tuberculous)

Mycobacterium: Acid Fast Bacilli

Standard staining met h ods

y Zie

hl-Neelsen (

hot staining)

y Kinyoun (cold staining)y Auramine o fluorescence

Z iehl Neelsen Stain

y Basic fuc h sin + ph enol (en h ance fuc h sin penetration into lipids)y Was h y Acid alco h ol (decolorizer)y Was h y Met h ylene Blue (Counter stain)

O ther Acid Fast O rganisms

y Norcardiay Actinomycesy Corynebacteriumy Rh odococcus

Culture

y M. Leprae HAS N O T BEENG RO WN O N CULTURE!y O th ers:

- Lowenstein Jensen (L J) medium- Middlebrook 7H9 (liquid)- Middlebrook 7H10 (Agar based)- Middlebrook 7H11 (Agar based too!)

y Clinical specimens : 7 days 6 weeks (Sometimes, even up to 12 weeks or more!)y As a w h ole, mycobacterium are VERY slow growing

Mycobacterium Acid Fast Bacilli

y Non-motiley Non-spore formingy Weakly G ram ( + )y Aerobic or microaerop h ilic (M. Tuberculosis is strictly aerobic)y Straig h t or slig h tly curved rod-s h apedy Some coccobacillary

- Filamentous- Branc h ed forms

y Some spp produced pigments (yellow orange)- In th e dark (scotoc h romogens)



- After exposure to lig h t (p h otoc h romogens)- No-pigmentation (Non-c h romogens)

y Some slow growers (>7/7 to form visible colonies)y Some rapid growers (<7/7)y 1959 Runyon divided mycobacterium into 4 groups

i. G

roup I =Ph

otoch

romogensii. G roup II = Scotoc h romogensiii. G roup III = Non-C h romogensiv. G roup IV = Rapid growers

y G roups I-III are slow growers

Mycobacterium:

y Can be isolated from a variety of sources- Clinical specimens- Environment (water, soil, dust)

y Can survive for long periods of timey As pat h ogens

i. O bligate pat h ogensa. M. tuberculosisb. M. bovisc. M. africanumd. M. ascaticume. M. farcinogensf. M. h aemop h ilum

g. M. lepraeh . M. malmoensei. M. microtij. M. paratuberculosisk. M. s h imodeil. M. Simiaem. M. Szulgai

ii. Facultative pat h ogensa. M. avium

b. M. ch elonaec. M. fortuitumd. M. kansasiie. M. ulceransf. M. terraeg. M. genavense

h . M. intracellulare

i. M. marinumj. M. senegalensek. M. scrofulaceuml. M. xenopim. M. malmoense

TU BERCULO SIS

y Ch ronic granulomatous diseasey Affects h umans and many mammalsy At one time single most important infectious disease of h umans ( 1/7 th of all deat h s

worldwide)y P resently:

- 1/3 world population infected- Cause about 3 million deat h s per year (>5% of all deat h s)- Is becoming a reemerging disease because of HIV

y Caused by 4 very closely related spp:



i. M. tuberculosis (Humans)ii. M. bovis (Cattle)iii. 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 many M. canetti (Very rare M. TB c smoot h )y M. bovis variant in good few TB in vets (M. caprae)

By th e way, t h is is a vole:

Mycobacterium T uberculosis

y Acid fast bacteriay O bligate aerobey Non-motiley Non-sporingy Unencapsulatedy Straig h t / Slig h tly curved rody 3 x 0.3 µm in sizey G row in several enric h ed media

LJ most widely used(Wh ole egg, glycerol, asparagine, salt)+ Malac h ite green (in h ibit contaminants kills ot h er microorganisms)

M. TB M. bovis M. africanumAtmosp h ericpreference

O bligate aerobe (onsurface)

Micro-aerop h ilic (fewmm below surface)

Micro-aerop h ilic

G rowt h in LJ Heaped up luxurianteugonic

Small flat dysgonic Intermediate

Reduce nitrate Nitrite

+ - Variable

Niacin production ++ + /- + /-Sensitivity topyrazinamide

S R S

Sensitivity to t h iopen-2 R S S





y If part of en tr y m ou th (m ilk, food)= Prim ar y com ple y To n sily C ervical lym ph n od e en lar ged (Scrof u la)y In tes tin e (ileoca ecal r egion + m ese n teric lym ph n od e)

y If part of en tr y skin y Skin + region al lym ph n od es = Prim ar y com ple (Pro sp ec ter s

art)

1-2 wee ks

y Usu ally lim it pri m ar y in f ec tio n

Gra nu lom a wit h cen tral case atio n Q u iesce n t Fibrobla sts Scar + Calcificatio n

In itial l es ion (Ghon Focus)

Hilar ly m ph nod es en lar ges

O the r part s of lung s Hem ato genous Spread Lym phati c Spr ead

T-ce ll clones (An tigen spec ific)

Cytoki n es

M acrop hage activatio n

Gra nu lo m aFor m atio n

Cen tral Caseatio n

(Cheese like in app eara nce & consistency)



NO T ALLh osts can completely destroy mycobacterium Dormant (How??)

= immunocompromised Reactivation (?? Reinfection) P ost-primary TB

STAG ES O F PRIMARY TB IN CH ILDHOOD

Time from onset C h aracteristics3-6 weeks y P rimary complex develops

y TB Conversion + 2-6 mont h s y P rogressive h ealing of primary complex

y P ossibility of pleural effusion6-12 mont h s y P ossibility of miliary or meningeal TB1-3 years y P ossibility of bone or joint TB3-5 years y P ossibility of gastro-urinary or c h ronic TB

Just for fun Balita = Bawa h Lima Ta h un

PO ST PRIMARY TB

y Most of t h ose exposed to M. TB= P rimary complex h eals completelyy O nly evidence ( + ) = Tuberculin test ( + )

y In some, after mont h s/years/decades= Reactivation or ?? Exogenous infection P ost-primary TB- Larger local lesions- Minimal lymp h atic involvement- > open cases infective- Especially apical region of lungs

y Reactivation- Spontaneous- Decrease immune responsiveness

y P rocess of granuloma formation= Same except more extensive wit h large areas of caseation (tuberculomata)

y P roteases & Cytokines (e.g. TNF)y P rocess

= Erode wall of bronc h us Liquefied content Expectorated Aerobic (open cases) Bacilligrowt h

y In immunocompromised:

- Cavitation rare!- Lymph atic & h ematogenous spread common Cryptic disseminated TB

D iagnosis

y Clinicaly X-rayy Tuberculin test

- O ld tuberculin



- Pu rified prot e in derivati ve (PP )- M an to ux tes t In tra cu ta n eou sly - Heaf t es t Gun wit h 6 pro ng s - Tin e tes t Dried PPD on to pro ng s for 1 tes t (di spo sabl e)

PPD activities

- 1 100 100 iu }1 1000 10 iu } 0 1 m l1 10000 1 iu }Sta n dard : 10 iu iu = in te rn atio n al un it

(+ ) In du ratio n <5 mm

5-10 mm

>10 mm

y In terpr e tatio n - n de m ic ar eas

- on -e n dem ic ar eas - C h ildr en - Immun ocom pro m ise dy Lab spec im en s

= Spu tum , bro n ch ial wa sh ing s, biop sies , gastri c aspirat es , C SF

Pleu ral fl u id, e tc y M icro scop y y C u ltu re

If C SF or Pleu ral Flu id or Biop sy, n o n ee d to d eco n ta m in at e or cen trif ug e � Direc tly depo sit a n d i n ocu lat e �

Spu tumDec on ta m in at e wit h Na O h (4%)

C e n trif ug e

Depo sitInocu lat e on to cu ltu re

m edia (LJ or

m iddl ebrook' s)

Afte r 7 da ys,read ( g rowt h)



DRUG SUSCEP TIBILITY TESTING

y In L.J, containing doubling dilutions of drugs

TREATMENT

Drugs = 3 groups

1. Sterilizing- Rifampin- P yrazinamide

2. Bacteriostatic- Eth ionamide- P rot h ionamide- Th iacetazone- P -aminosalicylic acid- Cycloserine

3.

Bacteriocidal- Isoniazid- Streptomycin- Eth ambutol

Also: Multidrug treatment (MDT)

O ther ways to D iagnose TB

y -P G L (glycolipid)y P CRy BCG (Bacillus Calmette- G uerin)- P rotects c h ildren for severe TB- O ffers cross protection against Leprosy

Leprosy

(Morbus Hansen [MH])

(Hansen s disease)

Definition: A c h ronic mycobacterial disease primarily affecting t h e perip h eral nervous system andsecondarily involving skin and ot h er tissues.

History

600 B.C written records in India 150 B.C written records in C h ina 2nd century BC Egyptian mummies From Egypt -> Europe (150 AD: G reece) Th en -> Americas & ot h er parts of t h e world 1983: WHO -> Estimated total in world 10.5 million





(paucibacillary)

Intense granulomatous response -> Damagemajor nerves ( h ardening)

Bacilli ++++ (multicacillary)

Immune response greatly weakened (anergy)

Ear lones bacilli ++++

Th ose wit h th e better immune system will suffer from t h e tuberculoid form. Th ose wit h th e lepromatous form will h ave a negative tuberculin test. Th e safest area to wear earings/studs is at t h e ear lobes. Anyw h ere else, t h en it will easily

affect t h e ear cartilage. It could lead to cauliflower ear. ( P ak Nasa s story)More WHO criteria:

Borderline tuberculoid (BT)

Mid-Borderline (BB) Borderline Lepromatous (BL)

O th er signs & Symptoms

Eye

Uveitis

Corneal infection secondary to eyelid paralysis Th e 2 symptoms above mig h t lead t h e person to suffer from blindness

Bone resorbtion

Collapse of nasal bone Sh ortening of fingers & toes (autoamputation)

Neuritis

O rch itis

Immune-Complex Nep h ritis

Facial skin may become crumpled and look like a lion (Facies leonina)



DIAG NO SIS

History & p h ysical examination

Slit-skin smear and punc h 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 t h e Ch inese

Split-skin smear - Acid fast staining

Record # of bacteria/ h igh 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 w h en stains unevenly & weakly

Morphological Index (MI):

Percentage (%) of regularly stained (viable) Increase MI Active disease

MI decreases wit h ch emot h erapyO ther D iagnosis methods:

-P G L P olymerase C h ain Reaction ( P CR)

Treatment:

Dapsone (diaminop h enyl sulp h one DDS)

Before 1982 Standard monot h erapy for all formsDDS Resistance -> MDT

Add

Rifampin Clofazimine (side effect: Skin discolouration)

O th er drugs

P rot h ionamide O floxacin Minocycline

WHO

Paucibacillary

Rifampin Dapsone (G ive t h ese for 6 mont h s)

Multibacillary

Rifampin Dapsone Clofazimine (G iven for 2 years or more)

Reactions:

Lepra/Jopling s Type I:

Delayed Type Hypersensitivity Vasculitis (Type II HS)

Lepra/Jopling s Type II

Eryth ema Nodosum Leprosum

Infections by environmental ( O pportunistic) mycobacteria (P ak Nasa said t h is part is not important)



Runyon s classification

a. Ph otoc h romogens ( G roup I) M. Kansasii M. Marinum M. Simiae

b . Scotoc

hromogens (

Group II) M. Scrofulaceum

M. gordonae M. szulgai

c . Non-Ch romogens ( G roup III) M. avium M. intercellulare M. malmoense M. xenopi M. ulcerans M. terrae

d . Rapid G rowers ( G roup IV)

M. ch

elonaePrincipal types of opportunistic mycobacterial infections in man

Lymph adenopat h y

M. Avium complex M. Scrofulaceum

Skin lesions

a. P ost-trauma abscesses M. Ch elonae M. Fortuitum M. Terrae

b . Swimming P ool G ranuloma M. Marinum

c . Buruli ulcer M. Ulcerans

P ulmonary Disease

M. Avium complex M. Kansasii M. Xenopi M. Malmoeure

Disseminated Disease

a. AIDS related M. Avium complex M. G enevense

b . Non-AIDS related M. Avium complex M. Ch elonae

Documents

5. Mycobacterium