Download - [Micro] mycobacterium tuberculosis
‘’ THE WHITE PLAGUE OF EUROPE’’17 & 18 CENTURY
23/04/2013Updated may 2015
HISTORYNEOLITH AGESEGYPTIAN MUMMIESROBERT KOCH ISOLATED IN 1882SENITORIASTREPTOMYCIN 1946ISONIAZID 1952
Member of Mycobacterium tuberculosis Complex
1.M tuberculosis2.M bovis3.M BCG4.M ulcerans5.M africanum
MORPHOLOGYBACILLUS : rod shaped, non- spore bearing 2-4um *0.2-0.5 umObligate Aerobe, growth enhanced by CO2Slow growingCarbon compds; oxidation & energyHigh lipid content of wall hydrophobicResistant to oral fluids
MAJOR HEALTH PROBLEMWHO 2008:1/3 population effected8.8 million new infected cases1.6 million died in 2000Every second a case infectedBy end of 2014; 12.3 million smear positive cases & 150,000 MDR treated
WHO 2014 global reportt9 million developed tb;;6.1 million cases ; 5.7 new & 0.4 million already on TM
2014 report: 122 countries 56% SEA & pacific; ¼ Africa
India 24% & China 11%1.5 million died3,60000 had HIV tooON DECLINE:Diagnosis n therapy from 2000-2013MDR 2013; 3.5 %
PAKISTAN: GFATMFifth highest burden country with TBFourth highest burden with MDR tbWHO’s National Professional Officer for Tuberculosis Control Dr Ghulam Nabi Kazi pointed out that
2012 over 284,000 cases of Tb detected & put on tm
this year the figure would touch around 300,000.
around 420,000 new cases appeared every year.12 tertiary care hospitals involved in Pakistan in diagnosis & t/m
CELL STRUCTURE• Slow growing: generation time 15-20hrs• Intracellular parasite• CELL WALL: 60% lipids• 3 components: 1. MYCOLIC ACIDS: long chain FA C78- C90
Bound to proteins, polysaccharides• Alpha branched structure• 50% dry weight of cell envelope• Form hydrophobic shell around MTB
LIPID BENEFITSPERMEABILTY BARRIER FOR1. Stains & dyes2. resist antibiotics3. resist killing by acids & alkalis
4. resist osmotic lysis by complement
5. resist oxygen and survive intracellular killing by macrophages
VIRULENCENO ATTACK BY:• CATIONIC PROTEINS• LYSOZYME• OXYGEN IN PHAGOCYTIC GRANULES
EXTRACELLULAR: Complement not deposited
2.CORD FACTOR: abundant in virulent strains serpentine cords. parallel chains;
toxic to mammalian cells
inhibit PMN migraton
3 Wax D : Freund’s adjuvant
STAINING CHARACTER’SBASIC STAINS
Gram’s stain: not taken/weakly taken Ziehl Neelsen stain;Carbol fuchsin: not readily taken, pushed by heat
ACID FAST: once taken cannot be decolorized by alcohol. 95% alcohol + 3%HCL(ACID-ALCOHOL) decolourize all but mycobacteria
HOT ACIDS & Sonication: destroy acid fastness
Lipid effects on tissueMuramyl dipeptide + mycolic acid complex Granuloma formation
Phospholipds: Caseous necrosis:CORD FACTOR: trehalose 6,6’-dimycolate1. Inhibit migration of leukocytes 2. Chronic granulomas3. Immunologic adjuvantProteins: tuberculin reaction; elicit Anti bodies
CULTURE MEDIA(non/selective)Semisynthetic Agar medium.Middlebrook 7H10 & 11Contents: salts, cofactors + malachite green/ABglycerol albuminoleic acid catalase
Mjddlebrook 7H11; caesin hydrolysate as well
Large inocula needed in several weeksColonial morphology, sensitivity testing
INSPISSATED EGG MEDIALOWENSTEIN JENSEN MEDIA:Complex organic substances;
fresh egg, yolks. potato flour
SaltsGlycerolMalachite green; selectiveSmall innocula yield growth in 3-6 weeks
BROTH MEDIAMiddlebrook 7H9, 7H12Tweens: water -soluble esters of fatty acids
wet the surface, dispersed growth in liquid
Rapid growth PHYSICAL & CHEMICAL AGENTS;Resistant bacterium; hydrophobic nature, clumping
Pencillin, malachite green, acids, alkalis kill others
PATHOGENICITYM tuberculosisSusceptible ; Humans & guinae pigsResistant: fowl, cattle
M bovisSusceptible; cattle, humans
M KansasiiLesion like MTB
PATHOGENESISDroplet infection; 1-5um nuclei
family, hospital personnel. < 25umSpeak, cough, sneeze settle in alveoliCytokines, lymphokines stimulate monocytes, Primary infection of ALVEOLAR MACROPHAGES. Unrestrained intracellular proliferation in non immune alveolar macrophages
Lesions; 1-2 months in lungsHypersensitivity: polysaccharides
Disseminated infection via macrophages to LN& blood
In 3-8 weeks cell mediated immunity develops
Activated macrophages at infection sites
Granuloma formation
Ag-specific activation of CD4+ T cells, clonal expansion
IL2 secretion increased & IF -gamma
IL2 receptors expression increased
Activation of gamma interferon to kill M tuberculosis by macrophages
Population exposure; Asia, Africa, latin America
overcrowding, socioeconomic conditions medical care provisionGenetic predisposition HLA Bw15AgAge ; extremesMalnutrition, alcoholics, destituteCoexisting disease : diabetes, silicosis, AIDS
PATHOLOGYHOST FACTORSMTB factors….no; multiplication LESIONSExudative; like pneumonia Oedema, polys, mononuclears surround Mtb
Fate; 1 resolution LN; enlarge/ Gohn’s complex
2 necrosis TT; positive 3 .productive
GOHN’S COMPLEX L NODE; enlarged, Caseation, calcificationChildrenAdultsREACTIVATION; surviving Mtb multiply; APEX
chronic tissue lesion; tubercle, caseation. Fibrosis
LN: Less effect
REACTIVATIONLatent infection reactivatedPartial immunityMycobacterial Ag increased in tissues intense inflammatory response by monocytesType 4 hypersensitivity responseDense mononuclear infilterates cause tissue damage due to release of O2 radicles and neutral proteases
Tissue damage: Caseous necrosis followed by liquifactive necrosis in absence of treatment
PRODUCTIVE TYPEChronic Granuloma/tubercle; 3 zones1 central area: large multinucleate giant cells with Mtb; later caseous necrosis
2. Mid zone; Pale epithelliod cells; radially set
3 Peripheral zone; fibroblasts, lymphocytes, monoytes cells; later fibrosis
Tubercle; empty in bronchus; form cavity
heal by fibrosis & calcification
SPREADBlood; LN.tubercle erosion in vein
Lymphatics; LN; miliary tbBronchi… aspirated to lungsGIT; swallowed food, lung
INTRACELLULAR GROWTH. Resist chemothera1.MONOCYTES2.RES3.GIANT CELL
SYMPTOMSCOUGHSPUTUM… early tissue necrosisDysponea; late tissue necrosis ;
parenchymal damage of lung
Fever; IL1 systemic effects
Weight loss; TNF alpha; cachectin
X-ray: Apical cavitary lesion of lung
PPD positiveHITOPATHOLOGYGRANULOMASEpitheloid cells; activated macrophages
Giant cells; most successful host tissue response
Activated lympocytesTubercle/granulation tissue
TUBERCULIN TESTMATERIAL;OLDTUBERCULIN; 6 week growth in broth
Conc.filterate has tuberculoprotein
PPD; chemical fractionation of OT PPD-S; Siebert’s Lot no 49608 standardTU; Biological activity of sp.wt of PPD S
Dose; 1 TU, 5 TU, 250TUstandardize by comparative activity in humans
TUBERCULIN TEST5 TU O.I ml injected intracutaneousRead in 48-72 hrs10 mm or > Positive: 4-6 weeks of infectionOedema. induration, erythemaPersist many days; weak disappear Central necrosis may be seen
DIAGNOSISClinicalSpecimenSmearCultureMolecular probe Gene sequencing
Tuberculin testPositive: infection (4-6 weeks)/ or past BCG vaccinationNEGATIVE:No exposureAnergy;, AIDS, Hodgkins measles, immunosuppression,
sarcoidosis overwhelming TB,
LAB DIAGNOSISSputum: 3-5 morning specimen CULTURE; decontaminate/centrifugeEgg based medium; LJAgar & Broth based media:Middlebrook AFB; Z-N stainAuramine-rhodaminePCR; respiratory smear positive samples
NEW DIAGNOSTIC TECHNOLOGIES BACTEC RAPID RADIOMETRIC CULTURE SYSTEMDeveloped by Becton DickinsonLiquid medium with C14 labelled palmetic acid
Automated early detection growing Mycobacteria use acid and release C14
Growth detected in 12 daysExpensiveFacility should handle radioactive material
HIGH PERFORMANCE LIQUID CHROMATOGRAPHYREF LAB: SPECIATION of mycobacteriaMycolic acid profiles vary in speciesDNA detection:PCR; 99% specific, 55-90% sensitive
STRAIN; clinical/epidemilogical typing
gene seq 6110 targeted:
DNA frag generated by restriction endonuclease digestion,
separated by electrophoreses.
Probe against 6110 used
ID ID OF M TUBERCULOSIS CULTURES
1.CONFIRM BY ZN STAIN; difficult to emulsify; others easily emulsify2.PIGMENT: leave in light for 2hrs re-incubate at 370 C overnight non- chromogen in light/dark3. Incubation of s/c at 25 0 C; no growth4.Growth on LJ Medium with p Nitrobenzoic acid 500 ug/ml No growth
PCRDNA/RNA probes and amplification usedDNA & RNA sequences extracted from mycobacteria in sputum
EpensiveSpecialist training neededMYCODOT ANTI-BODY
20 minute immuno-assay
Pureified lipoarabinomannan; highly immunogenic
Detects AB in active disease ie high levels; not screening
BACTERIOPHAGE TEST FOR SPUTUMFAST PLAQUE ASSAY:Mix sputum with a reagent with bacteriophageMycophages infect mycobacteriaUse virucide to kill bacteria outside mycobacteria
Mycobacteriophages replicate inside mycobacteria, cause their lysis and released out
Other rapid growing non pathogenic mycobacteria are addded,infected by these phages.
Sample is incorporated in agar mixture, plated and incubated overnight at 37 0 c
Phages lyse mycobacteria, leaving zones of clearing ie holes
Thi proves pt sample had M TBsensitivity like culture: can detect 100 tubercle bacilli/ml
GLOBAL TB REPORT 201415 vaccines under trial10anti tb drugs undergoing clinical trials
4 month versus 6 month therapy duration2 approved delamanidBedaquilineTARGETS: 203595% reduction in deaths90% reduction in incidence
GAMMA INTERFERON RELEASE ASSAYMTB specific Ag: (not BCG/non myco tb) ESAT 6 CFP 10CD4 cells release gamma interferon in whole blood
ELISA ASSAYQuantiferon GoldT spot TB ; mononuclear cells used
conversionTo negative:Isoniazid T/MBCG VACCINE; + 3-7 yrs - elimination of viable Mtb
HEALTHY; after yrs negative retest: 2 weeks positive by boost inj
Molecular probes1 day to detect/ 2hrsRapid, Sensitive, SpecificMycobacterium 10,000 rRNA copies /cell; natural amplification, easy detection
Hybridization of DNA with rRNA sequences single strands separated from hybrids
DNA probes attached to chemicals that chemiluminescence in hybrids & measure by lumino- meter & equivalent to amount of hybrid
Probes for M tub complex & MAC
TREATMENTFIRST LINE DRUGSPRIMARY: daily 1—2 months; 9 biweeklyIsoniazidRifampinOTHERS:PyrazinmideEthambutalStreptomycin6 months regime ; 4 drugs 2 months; iso/rif biweekly
SECOND LINE DRUGSTOXIC; Used in T/M failure with first-line
KENAMYCINCAPREOMYCINETHIONAMIDECYCLOSERINEOFLOXACINCIPROFLOXACIN
DRUG RESISTANCEOne in 106 or 108 mutate spontaneouslySingle drugFirst line drugsCombinations used: 4 drug regimeIsoniazid, rifampine, pyrazinamide, ethambutal
Second line; toxic, less effective, bothReserved for special circumstances; t/m failure, MDR
riskfactors: HIV, Asian, Latin American migrants, MDR area,
Mechanisms of drug resistanceIsoniazid resistance:katG; deletion, mutation in catalase-peroxidase gene
no or less catalase functionInhA gene: enzyme encoding mycolic acid synthesis
Streptomycin resistance: mutation in genes coding
Ribosomal S12 protein, 16SrRNA, rpsl, rrs
MDR: isoniazid & rifampin resistance problem in T/M & control hospitals, prisons, HIV t/m of contacts after sensitivity test , 3 or > drugs
XDR: extensive drug resistance WHO defination R : INZ & PYR flouroquinolone
3 second line injectables
CAUSES 1. Poor infection control2. Ineffective T/M3. Diagnostics poorFATE:PoorMortality; 64%
FUNDS8 billion US dollars/ year needed for global full response to TB epidemic
2/3 for detection & tm of drug susceptible tb
20% MDR T/M10% rapid tests & diagnostics5% HIV associated tb activity
PREVENTION & CONTROL1.Public health control: t/m cases: prompt & effective with follow up
Contacts: diagnose{ tt, x ray} & treat 2.Asymptomatic cases: tt + Treat with drugs: children, immunosuppressed3. Host resistance of person: reactivate or convert asymptomatic to disease
Malnutrition, gastrectomy, infection, HIV, steroids4. BCG 5. Cattle eradication 6. milk pasteurization
Atypical mycobacteria
ANONYMOUS MYCOBACTERIAENVIRONMENTAL MYCOBACTERIAMYCOBACTERIA OTHER THAN TUBERCLE; MOTTFound in soil, waterCause opportunistic infection in HIV/Immunosuppressed
DIAGNOSTIC FEATURESCHROMOGENSSCOTOCHROMOGENS; Pigment in darkPHOTOCHROMOGENS; Pigment in lightNON-CHROMOGENSTEMPERATURE:Grow at 25 0 CGrow in presence of P nitrobenzoic acidGROWTHSlow growers; 2 rapid growers
Pul inf S/R 25 oC Pigm PNBMAC /MAI S + N/S +
M kansasi S + N/S +
M xenopi S + N/S +
M malmoense S + N +
M scrofulaceum S + S +
M chelonae R + N +
M fortuitum R + N +
TRADITIONAL RUNYON Classification of MYCOBACTETB COMPLEX M tuberculosis, M africanum, M bovis PHOTOCH; M kansassi,marinum, simiae,asiaticumSCOTOC; M gordonae,scrofulaceum,szulgi, flavescens
NON CHROM; MAC, Ulcerans, xenopi, trivale, terrae, malmoense,gastri, genavense, hemophilium, celatum
RAPID GROWERSM abscessus M phlei M immunogenum
M fortuitum M smegmatis M vaccae
M chelonae M mucogenicum
MAC MAIGrow optimally at 41 CSmooth, soft non pigmented colonies,Ubiqutous; water, soil, food, animals, birdsImmuno-competent humans affectedAIDS: Opportunistic esp <100/ul25-50% pts develop bacteremia, disseminated infection. Decrease with HAART and azithromycin
Tissue infilteration…organ dysfunction eg lung,skin, soft tissue, LN, bone, CNS
C/O fever,night sweat,abd pain, diarrhea, wt loss
M kansasiiPhotochromogen, complex media at 37CPulmonary and systemic disease like TB esp ICompro
t/m; Rifampin, ethambutol,isoniazidM scrofolacium; scotochromogen, waterAdults with chronic lung disease lymphadenitis in childrenM marinum/ulcerans: water at 31 C, fish infected; swimming pool granulomas
M chelonae-abscessus; rapid grower; skin, bone. soft tissue infection,rep tract, cystic fibrosis