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  • 1. An overview and Updates in TBByGamal Rabie Agmy , MD , FCCPProfessor of Chest Diseases ,Assiut UniversityERS National Delegate of Egypt
  • 2. *The increasing prevalence oftuberculosis in both immunocompetentand immunocompromised individualsin recent years makes this disease atopic of universal concern.Introduction
  • 3. *The World Health Organization (WHO)estimates that each year more than8 million new cases of tuberculosis occurand approximately 3 million persons diefrom the disease.*Ninety-five percent of tuberculosis casesoccur in developing countries.*It is estimated that between 19 and 43% ofthe worlds population is infected withMycobacterium tuberculosis, thebacterium that causes tuberculosisinfection and diseaseEpidemiology
  • 4. Epidemiology Most cases in the US are due toreactivation, especially amongst immigrants Highest risk of progression to active TB iswithin 2 years of seroconversion Increase in incidence in late 1980s-early 90slargely due to HIV Needs to be reported to the healthdepartment
  • 5. Microbiology Aerobic Bacillus (rod-shaped) Non-spore forming Non-motile Cell wall – mycolic acid – retains acid faststain Growth - doubling time of 15-20 hrs. 3-8 weeks for growth on solid media
  • 6. TB Skin Testing PPD – purified protein derivative oftuberculin (antigenic) Delayed type hypersensitivity reaction PPD may not become “positive” until 3months after exposure Boosting effect
  • 7. Skin Test Interpretation PPD >/= 5 mm:– HIV patients– Recent contacts of someone with TB– Fibrotic changes on CXR c/w prior TB– Organ transplant recipients– Immunosuppressed (includes patientsreceiving the equivalent of 15 mg/day ormore of prednisone for one month ormore)
  • 8. Skin Test Interpretation PPD >/= 10 mm:– Recent immigrants (< 5 years) from highprevalence areas (Eastern Europe, LatinAmerica, Asia, Africa)– IV drug users– Residents and employees of high risk facilities(hospitals, nursing homes, homeless shelters,prisons)– Children < 4 years of age– Mycobacteriology lab personnel
  • 9. Skin Test Interpretation PPD >/= 10 mm:– People with medical conditions that placethem at high risk for active TB Chronic renal failure Diabetes mellitus Silicosis Leukemias/lymphomas Carcinoma of the head/neck or lung Weight loss > 10% of ideal body weight Gastrectomy/jejunoileal bypass
  • 10. Skin Test Interpretation PPD >/= 15 mm:– Low risk people– Routine tuberculin testing notrecommended for low risk populations
  • 11. Skin Test Intrepretation False positives:– Non-tuberculous mycobacterial infection– BCG vaccination False negatives:– HIV– Malnutrition– Steroid therapy– Recent infection
  • 12. BCG Bacille Calmette-Guerin vaccination: Live attenuated mycobacterial strain derivedfrom M. bovis Can yield false positives to PPD – less likelyas time from vaccination increases Reactions > 20 mm likely are true CDC advises that the PPD be interpreted bythe same guidelines (ignore the BCG history)
  • 13. Quantiferon Testing Whole blood in vitro test:– Lymphocytes release IFN gamma inpresence of 2 TB antigens Will be positive in latent or active TB Advantages:– No error in interpretation– No follow-up in 48-72 hours– No boosting– Not affected by BCG
  • 14. Quantiferon Testing Disadvantages:– Must be processed within 12 hours ofcollection– False + with atypical mycobacteria– Too many indeterminate results withcurrent version (Q-Gold)– May be less reliable in pregnant women,children, and immunocompromised– Does not distinguish between active andlatent TB
  • 15. Causative organism;Mycobacterium tuberculosis COMPLEXStained with:-Modified gram stain: gram positive.-Carbolfuchsin stain: *Cold method(Kynon)*Hot(Zeil-Neelson)- Fluorescent dyes: rhodamine andauramine stains.Bacteriology
  • 16. QUANTITATION SCALE FOR ACID-FAST BACILLUSSMEARS ACCORDING TO STAIN USEDCarbolfuchsin (× 1,000)Fluorochrome(× 250)Quantity ReportedNo AFB/300 fields No AFB/30 fields No AFB seen1-2 AFB/300 fields 1-2 AFB/30 fields Doubtful, repeattest1-9 AFB/100 fields 1-9 AFB/10 fields Rare (1+)1-9 AFB/10 fields 1-9 AFB/field Few (2+)1-9 AFB/field 10-90 AFB/field Moderate (3+)> 9 AFB/field > 90 AFB/field Numerous (4+)
  • 17. Zeil-Neelson StainingWire 0.01 ml of specimen 200mm2 slideOil immersion field 0.02mmSlide=10000 field=0.01ml specimen10,000 organism/slide=1 AFB/field=1000,000 organism/ml1000 organism/slide=1 AFB/10 field=100,000 organism/ml100 organism/slide=1 AFB/100field=10,000 organism/ml
  • 18. QUANTITATION SCALE FOR ACID-FAST BACILLUSSMEARS ACCORDING TO STAIN USEDCarbolfuchsin (× 1,000)Fluorochrome(× 250)Quantity ReportedNo AFB/300 fields No AFB/30 fields No AFB seen1-2 AFB/300 fields 1-2 AFB/30 fields Doubtful, repeattest1-9 AFB/100 fields 1-9 AFB/10 fields Rare (1+)1-9 AFB/10 fields 1-9 AFB/field Few (2+)1-9 AFB/field 10-90 AFB/field Moderate (3+)> 9 AFB/field > 90 AFB/field Numerous (4+)
  • 19. Cultures:- Lowenstein Jensen media: 6-8weeks.-Bactec media: 2-8days. Radiolabelled 14clabelled palmitic acid-Mycobacterial growth indicator tube:Middbrook broth+o2 sensitive fluroscent sensor toindicate growth& bacilli can be identified by GenProbe method at the same day of detection.
  • 20. Diagnosis of Active TB Acid fast stain of sputum Sputum AFB culture (culture neededfor drug susceptibility) Radiographic imaging (CXR, CT) PCR/NAT Fluid Aspiration Tissue biopsy – higher yield than fluid
  • 21. Direct Methods1-Direct Microscopy (ZN, Kinyoun,Flurochrome).2-Culture (Traditional, Rapid methods).3- Detection of DNA or RNA ofmycobacterial origin( PCR, LAMP, TAA / NAA, LCR, FastPlaque).
  • 22. Direct MicroscopicExamination*Hallmark of staining is Ziehl-Neelsen stainedslides.Easiest & quickest diagnostic test.* Limited sensitivity (46-78%) but specificity isvirtually 100%.* Centrifugation & flurochrome staining(auramine O)with UV microscopy markedlyincrease the sensitivity & a large number canbe examined in a much shorter time.**Chest 1969;95:1193
  • 23. Direct MicroscopicExamination􀂄 ZN staining requires = 105bacilli/ml.􀂄 TB bacilli appear asstraight/curved rods (1-4μ x0.2-0.8μ) singly, in pairs or inclumps.􀂄 The yield of microscopicexamination correlates wellwith the extent of disease, thepresence of cavitation, and thequality of specimen.􀂄 It is a good marker forinfectiousness & the responseto the treatment.
  • 24. Several approaches are being made toenhance thesensitivity of the smear microscopy :􀂄 Concentration of sputum sample by centrifugationenhances sensitivity to almost 100%.*􀂄 Treatment of sputum samples with Zwitterionicdetergent, also known as C18carboxyprophylbetaine(CB18) interferes with the innatebuoyancy of the bacilli and enhances the result ofsputum microscopy.***J Clin Microbiol 1999;31: 2371**J Clin Microbiol 1998;36: 1965
  • 25. Traditional Culture􀂄 More sensitive & can be positive even whenbacterial load is low(10-100 bacilli/ml).􀂄 Sensitivity 80-85%; Specificity 98%.􀂄 Required for precise identification of causativeorganisms.􀂄 3 Types of media are used:􀂄 Egg based: LJ, Petragnani and ATS.􀂄 Agar based: Middlebrook 7H10 or 7H11.􀂄Liquid based: Kirschner’s, Middlebrook 7H9.􀂄 Growth is slow and takes 6-8 weeks . There afterthe same length oftime is required for complete identification & sensitivitytesting.
  • 26. Broth Based Rapid CultureMethods􀂄 Micro colony detection on solid media.􀂄Radiometric (BACTEC).􀂄Septicheck AFB.􀂄 Mycobacterial growth indicator tubes(MGIT).􀂄 Substantial improvement in time todetection & totalnumber of positive cultures can be realizedfrom usingbroth based systems.
  • 27. Micro colony Detection on SolidMedia􀂄 Plates poured with thin layer ofMiddlebrook 7H11agar medium are incubated and examinedmicroscopically on alternate days for thefirst 2 daysand less frequently thereafter.􀂄 In less than 7 days micro-colonies ofslow growingmycobacteria such as M.tb can bedetected.
  • 28. Micro colony Detection on SolidMedia􀂄 Plates poured with thin layer ofMiddlebrook 7H11agar medium are incubated and examinedmicroscopically on alternate days for thefirst 2 daysand less frequently thereafter.􀂄 In less than 7 days micro-colonies ofslow growingmycobacteria such as M.tb can bedetected.
  • 29. BACTEC􀂄Growth is ascertainedby liberation of 14CO2as metabolized bymycobacteria &detected by BACTEC460 instrument &reported in terms ofgrowth index (GI) value
  • 30. BACTEC􀂄 Average time to recovery of M.tb fromsmear positivespecimens is 8 days.􀂄 When smear negative, culture positivesamples areexamined, mean time for detection is 14days.􀂄 More sensitive than traditionalmethod.*􀂄 Can also be used for drugsusceptibility testing.*J Clin Microbiol 1994;32: 918-925
  • 31. BACTEC􀂄 A special procedure unique toBACTEC system foridentification of M.tb complex is based onobservationthat p-nitro-α-acetylamino-β-hydroxypropiophenone(NAP) will inhibit organisms belonging toM.tb complexwhile having little or no effects on othermycobacteria.􀂄Drawbacks :􀂄Cost.􀂄 Problem of disposal of radioactivewaste.
  • 32. Septicheck AFB􀂄 Combines broth & solid media into asingle device(biphasic culture approach).􀂄 Contains 30ml of modified Middlebrook7H9 broth in CO2enriched culture bottle & a peddle withagar media- oneside of peddle covered with Middlebrook7H11; otherside contains Middle brook 7H11 with NAP.􀂄 Requires 3 weeks of incubation􀂄 Advantage: Simultaneous detection ofMtb. NTM, otherrespiratory pathogen & even contaminant.
  • 33. Mycobacterial Growth IndicatorTube (MGIT)􀂄Rapid Method.􀂄 Consists of round bottom tubescontaining 4 ml ofmodified Middlebrook 7H9 broth which hasan oxygensensitive fluroscent sensor at the bottom.*􀂄 When mycobacteria grow, they depletethe dissolvedoxygen in the broth & allow the indicator tofluorescebrightly in a 365nm UV light.* J Clin Microbiol 1999;37: 748-752
  • 34. Mycobacterial Growth IndicatorTube (MGIT)􀂄 Positive signals are obtained in 10-12days.􀂄 MGIT can also be used as a rapidmethod for thedetection of drug resistant strains of Mtbdirectly fromacid-fast smear positive samples as well asfrom indirectdrug susceptibility studies.􀂄Advantages over BACTEC􀂄Cheaper.􀂄 No problem of radioactive wastedisposal.J Clin Microbiol 1999;37: 45-48
  • 35. Detection and identification of mycobacteriadirectlyfrom clinical samples􀂄Genotypic Methods :􀂄PCR􀂄LAMP􀂄TMA / NAA􀂄Ligase chain reaction􀂄Phenotypic Methods :􀂄FAST Plaque TB
  • 36. Polymerase Chain Reaction(PCR)􀂄 Essentially PCR is a way to makemillions of identicalcopies of a specific DNA sequence , whichmay be agene, or a part of a gene, or simply astretch ofnucleotides with a known DNA sequence,thefunction of which may be unknown.􀂄 A specimen that may contain the DNAsequence ofinterest is heated to denature doublestranded DNA.
  • 37. Polymerase Chain Reaction(PCR)􀂄 Specific synthetic oligonucleotideprimers bind to theunique DNA sequences of interest and aheat stableDNA polymerase (Thermus aquaticus)extends theprimer to create a complete &complimentary strandof DNA.􀂄 This process is repeated sequentially25-40 times,thereby creating millions of copies of targetsequence.
  • 38. PolymeraseChainReaction(PCR)􀂄65Kd antigen (HSPs):􀂄Used earlier􀂄 Heat shock protein believed to bedistinct fromother bacterial HSPs.􀂄 This gene is identical in all species ofmycobacteria.􀂄 Therefore unsuitable for detecting M.tb,particularly in areas where species likeM.aviumor M.kansasii are prevalent.
  • 39. IS6110 :􀂄 It is a transposon which areself replicating stretches ofDNA.􀂄Function not known.􀂄 This sequence has been found in theM.tb complexorganisms (M.tb, M.africanum, M.microti,M.bovis).􀂄 IS6110 sequence generally occurs onlyonce in M.bovisbut is found as often as 20 times in certainstrains of M.tb,thus offering multiple targets foramplification.
  • 40. Polymerase Chain Reaction(PCR)􀂄 With recent modification PCR candetect even a fractionof a bacilli.􀂄Role in pulmonary TB :􀂄 Detects nearly all smear +ve andculture +ve cases.􀂄 Useful technology for rapid diagnosis ofsmear –ve casesof active TB.􀂄 Able to identify 50-60% of smear -vecases; this wouldreduce the need for more invasiveapproaches to smear -ve cases.
  • 41. Distinguish M.tb from NTM in smear +vecases asIS6110 sequence is not found in NTM.*􀂄 Should not be used to replace sputummicroscopy.􀂄 Sensitivity, specificity, & PPV for PCR is83.5%,99% & 94.2% respectively.***Am Rev Respir Dis 1991; 144:1160** J Clin Microbiol 1999;31: 2049-2055
  • 42. PolymeraseChainReaction(PCR)􀂄 Role in Extrapulmonary TB􀂄Limited Role􀂄 No comprehensive large seriescomparing theyield of PCR with other availableapproaches hasbeen published.􀂄 But at present, it is valuable adjunct inthediagnosis of TBM, pleurisy, pericardial TB& othercondition in which yield of other tests arelow.
  • 43. PolymeraseChainReaction(PCR)􀂄Disadvantages :􀂄 Very high degree of quality controlrequired.􀂄 Variation from lab to lab remainsignificant.􀂄 In pts. on ATT, PCR should not be usedas anindicator of infectivity as this assay remains+vefor a greater time than do cultures.*Am J Respir Crit Care Med 1997;155: 1804-1854
  • 44. High false +ve results in patients previouslytreated with ATT in contacts of sputum +veactivecases.􀂄High Cost.􀂄 So, better understanding of how to usethesetests in conjunction with available clinicalinformation is essential.**Thorax 1992;47:690-694
  • 45. LAMP*􀂄Loop-mediated isothermalamplification.􀂄 It is a novel nucleic acid amplificationmethod in whichreagents react under isothermal conditionswith highspecificity, efficiency, and rapidity.􀂄 LAMP is used for detection of M.tbcomplex, M.avium,and M.intracellulare directly from sputumspecimens aswell as for detection of culture isolatesgrown in a liquidmedium (MGIT) or on a solid medium(Ogawa’smedium).*Iwamoto T et al J Clin Microbiol 2003;41 :2616-2619
  • 46. LAMP􀂄 This method employs a DNApolymerase and a set offour specially designed primers thatrecognize a total ofsix distinct sequences on the target DNA.􀂄 Species-specific primers weredesigned by targeting thegyrB gene.􀂄 Simple procedure, starting with themixing of all reagentsin a single tube, followed by an isothermalreactionduring which the reaction mixture is held at63°C.􀂄60-min incubation time.
  • 47. LAMP􀂄 Due to its easy operation withoutsophisticatedequipment, it will be simple enough to usein:􀂄Small-scale hospitals,􀂄Primary care facilities􀂄 Clinical laboratories in developingcountries.􀂄Difficulties :􀂄Sample preparation􀂄Nucleic acid extraction􀂄Cross-contamination.
  • 48. TMA / NAA􀂄Transcription Mediated Amplification(TMA).􀂄 Nucleic Acid Amplification (NAA).􀂄 These techniques use chemical ratherthan biologicalamplification to produce nucleic acid.􀂄 Test results within few hours.􀂄 Currently used only for respiratoryspecimens.
  • 49. Ligase Chain Reaction􀂄 It is a variant of PCR, in which a pair ofoligonucleotides are made to bind to one ofthe DNAtarget strands, so that they are adjacent toeachother.􀂄 A second pair of oligonucleotides isdesigned tohybridize to the same regions on thecomplementaryDNA.
  • 50. Ligase Chain Reaction􀂄 The action of DNA polymerase andligase in thepresence of nucleotides results in the gapbetweenadjacent primers being filled withappropriatenucleotides and ligation of primers.􀂄 It is mainly being used for respiratorysamples, andhas a high overall specificity and sensitivityfor smear+ve and –ve specimens.
  • 51. FAST Plaque TB􀂄 It is an original phage based test.􀂄 It uses the mycobacteriophage todetect the presence ofM.tb directly from sputum specimens.􀂄 It is a rapid, manual test, easy toperform and has ahigher sensitivity than microscopy, in newlydiagnosedsmear +ve pts.**Int J Tuberc Lung Dis 1998;2: 160
  • 52. Indirect Methods􀂄Antibody detection :􀂄TB STAT-PAK􀂄ELISA􀂄India test TB􀂄Antigen detection :􀂄 TB MPB 64 patch test.􀂄Quantiferon-GOLD test.􀂄Biochemical Assays :(ADA, BromidePartition, GasChromatography).
  • 53. TB STAT-PAK􀂄Immuno-chromatographic test.􀂄 Has been evolved with a capability todifferentiatebetween active or dormant TB infection inwhole blood,plasma or serum.􀂄 Its value in in disease endemiccountries is yet to beascertained.**Eur Resp J 1995;8: 676
  • 54. Antibody detection by ELISA.􀂄 Several serodiagnostic tests,principally those usingELISA methodology for measurement ofIgG Ab areavailable.􀂄 38-Kd Ag provides serodiagnostic testwith mostfavorable test characteristics described,but is limited bythe lack of purified Ag.􀂄 Serum IgG Ab are observed to riseduring the first 3months of therapy but fall after 12-16months.
  • 55. Antibody detectionby ELISA.􀂄 Other purified antigens to whichantibodies are detected:􀂄 30 Kd protein antigen􀂄 16 Kd heat-shock antigen􀂄Lipoarabinomannan(LAM) – LAM is acomplexglycolipid associated with cell wall ofmycobacteria &is produced insubstantial quantities by growingM.tb.􀂄A60 antigen􀂄ES31/41 antigen
  • 56. Antibody detection by ELISA.􀂄 IgM Ab levels have usually been foundto be so low thattheir reliable measurement has beendifficult.􀂄 Serodiagnosis with crude Ag gives highfalse positiveresults.􀂄 These tests lack specificity becausepolyclonal Ab areused.􀂄 Use of monoclonal antibodies haveincreased theirspecificity.
  • 57. Antibody detection by ELISA.􀂄 It takes several months after diagnosisfor patients withpulmonary TB to reach maximum antibodytiters so thatserodiagnosis appears to be more useful inchronicextrapulmonary disease (bone or joint)than in acuteforms (miliary, TBM).􀂄 Serodiagnosis also has limited utility insmear negativepatients with minimal PTB; In pediatric TB& in diseaseendemic countries with high infection rates.
  • 58. Antibody detection by ELISA.􀂄 ELISA also has limited diagnosticpotential in AIDSprevalent population.*􀂄 Tests are expensive, require trainedpersonnel &difficulty in distinguishing Mtb & NTM.􀂄 Serologic tests have not yetdemonstrated sufficientperformance to warrant routine use incontrol programs.* Int J Tuberc Lung Dis 2000;4132: 5152-5388
  • 59. Antibody detection by ELISA.􀂄 Sensitivity and specificity of ELISAserodiagnostic testsusing measurement of serum IgG Ab toselectedmycobacterial Ag:Antigen Sensitivity Specificity38 Kd 49-89 98-10030 Kd 62-72 97-10016 Kd 24-71 97-99LAM 26-81 92-100A60 71-100 71-95
  • 60. Antibody detection by ELISA.􀂄 The detection of mycobacterialantigens byimmunoassay in clinical specimens withhigh & variableprotein content is difficult.􀂄 Detection in sputum presents evengreater clinicalproblem because sputum is a non-homogenous gel .􀂄 False positive rates are high.􀂄 Abandonment of this diagnostic tool.
  • 61. Insta test TB􀂄 It is a rapid in vitro assay for thedetection of antibody inactive TB disease using whole blood orserum.􀂄 The test employs an Ab binding proteinconjugated to acolloidal gold particle and a uniquecombination of TBAgs immobilized on the membrane.**Tuberc. Lung Dis 1998;2: 541
  • 62. TB MPB 64 patch test􀂄 MPB 64 is a specific mycobacterialantigen for M.tbcomplex.􀂄 This test becomes +ve in 3-4 daysafter patch applicationand lasts for a week.􀂄Specificity~100%, Sensitivity~98.1%.*􀂄 This promising test has been reportedso far only in onesetting in Philippines and needs to becarried out in othersettings.*Ind J Tuberc Lung Dis 1998;2: 541
  • 63. Quantiferon-GOLD􀂄 Due to advances in molecular biologyand genomics, analternative has emerged for the first time inthe form of anew class of in vitro assays that measureinterferon(IFN-γ) released by sensitized T cells afterstimulation byM. tuberculosis antigens.􀂄 Measures immune reactivity toM.tb.
  • 64. Quantiferon-GOLD􀂄 Interferon-γ assays measure cell-mediated immunityby quantifying IFN-γ released fromsensitized T cellsin whole blood/PBMCs incubated with TBantigens.
  • 65. QuantiFERON-TB ® test (Cellestis,Australia– Commercially available.– Measures amount of IFN-γ produced.(ELISA)– FDA-approved for the detection of LTBI,2001.􀂄ELISPOT assay (Oxford, UK)– Similar to QFT.– Measures number of reactivelymphocytes.– Not commercially available.
  • 66. Early assays employed PPD (samespecificity problemsas the TST).􀂄 Newer assays (e.g., QFT-Gold) employTB-specificantigens: ESAT-6 and CFP-10.􀂄 Proteins encoded within the region ofdifference 1 ofM.tuberculosis.􀂄 Not shared with the BCG sub-strainsand most NTM(except: M. kansasii, M. szulgai, M.marinum and nonpathogenicM.bovis).Quantiferon-GOLD
  • 67. Improved specificity: able to distinguishbetween TB andNTM, BCG infection.􀂄 Studies in contacts, HIV infected andchildren underway.􀂄 Recommended for use in “ALLcircumstances in which thetuberculin skin test is currently used”.*􀂄 Includes contact investigations,immigrant evaluation,surveillance (e.g. healthcare workers).*Mazurek et al MMWR 2005;54:15Quantiferon-GOLD
  • 68. IGRAs Vs. TST􀂄TST􀂄In vivo􀂄Single antigen􀂄Boosting􀂄2patient visits􀂄Inter-reader variability􀂄 Results in 2-3 days􀂄 Read in 48-72 hrs􀂄IGRAs􀂄In vitro􀂄Multiple antigens􀂄No boosting􀂄1patient visit􀂄Minimal inter-readervariability􀂄 Results in 1 day􀂄 Stimulate w/i 12 hrs
  • 69. IGRAs Vs. TST􀂄 QFT-g vs. TST Agreement = 83.6%*􀂄 Factors associated with discordance :– Prior BCG– Non-tuberculous mycobcateria immunereactivity– Site bias in reading TST– TB Treatment*Mazurek et al JAMA 2001;286:1740
  • 70. Biochemical markers ofDiagnosis􀂄Adenosine deaminase. (ADA)􀂄Bromide partition test.􀂄 Gas chromatography of mycobacterialfatty acids(Tuberculostearic acid).
  • 71. Adenosine Deaminase (ADA)􀂄 It is an enzyme of purine metabolism.The level of thisenzyme is 10 times higher in lymphocytes(T cells >Bcells) than in RBC.􀂄 Whenever there is cell mediatedimmune response to anantigenic stimuli, the ADA levels are thehighest.􀂄 ADA is measured by the colorimetricmethod of Giusti.
  • 72. enzymatic reaction is:Adenosine + H2O + ADA = Inosine + NH3+ADA􀂄 The amount of ammonia liberatedis measured bythe colorimetric method.Cut-off Sensitivity SpecificityPleural Fluid 50 IU/ml 95% 100%Ascitic Fliud 32.3 IU/ml 89% 98%CSF 9 IU/ml 100% 100%
  • 73. Bromide Partition Test􀂄 The partition of bromide ion betweenserum and CSFafter a loading dose reflects the integrity ofthe bloodbrain barrier.􀂄 Either by direct chemical measurementor by using anisotopic tracer, the ratio of bromide inserum to that inCSF can be estimated.􀂄 Values <1.6 are characteristic of TBM.
  • 74. In different studies the sensitivity andspecificity of thistest has been found to be near 90%.*􀂄 It may be false +ve in herpes simplex,listeria, mumps,measles, pyogenic meningitis andhypothyroidism.􀂄 With the availability of better tests, thistest has beengiven up.*Taylor J et al. J Clin Microbiol 1999; 34: 56-59
  • 75. Tuberculostearic Acid (TBSA)􀂄 TBSA is found in the cell wall ofmycobacterium.􀂄 It is identified by gas chromatographyor massspectrophotometry.􀂄 It is a costly investigation and requirescomplexanalytical equipment. (Seldom used)􀂄Sensitivity >95%,Specificity>99%.**French M et al. J Clin Microbiol 1998; 54: 987-990
  • 76. CT Scan and MRI Scan in thediagnosis of TB􀂄 The advent of CT and MRI imaging inthe last twodecades has redefined the approach inanalysis ofvarious diseases including TB.*􀂄 CT and MRI have shown severaladvantages overconventional radiology in early diagnosisand follow-upof TB in different parts of the body.*Buxi TBS Indian J Pediatr 2002;69:965-972
  • 77. Pulmonary TB :􀂄Lobar Pneumonia􀂄 CT is superior than plain CXR in pickingup theconsolidation, atelectasis and the hilar LNtherebymaking the diagnosis easy.􀂄 MRI reveals some of these changes,however, CT isthe diagnostic modality of choice in suchcases.􀂄Bronchopneumonia􀂄 On CT it is usually B/L and widespread,not alwayssymmetrical involvement of lungs.
  • 78. Hilar and MediastinalLymphadenopathy􀂄 CT and MRI depict the hilar andmediastinal LNequally well.􀂄 Calcification in the nodes is howeverbetter seen onCT.􀂄 Necrosis is seen as focal areas of lowattenuation ona CECT.􀂄 On MRI focal necrosis is seen as areasof increasedsignal intensity on T2W images.􀂄EBTB􀂄 HRCT is sensitive in the detection ofearlyendobronchial spread of disease.
  • 79. Miliary TB􀂄 Earliest form of miliary TB is detectableon HRCT.􀂄 Coalescing nodules result into patchyirregularopacities and HRCT shows this variationeffectivelyand has been described as “snowstormappearance”.􀂄 HRCT shows cavitation, which is notevident on plainCXR.􀂄Pleural Effusion􀂄 CT is sensitive to diagnose and defineeven minimalpleural effusion/pleural calcification.􀂄 Pleural fluid is seen on inversionrecovery MRimages as areas of increased signalintensity alongthe inner aspects of the chest wall.
  • 80. Skeletal TB􀂄Pott’s Disease (vertebral TB)􀂄 CT and MRI helps in demonstrating asmall focus ofvertebral body involvement and definingthe extent ofthe disease.􀂄 CT/MRI help to evaluate TB involvingthe craniovertebraljunction, sacro-iliac joint and posteriorappendages.􀂄 They are also helpful in assessment ofspinal canalencroachment , posterior elementinvolvement and indeciding the surgical approach.
  • 81. GIT TB􀂄 Strictures of the small bowel, mucosaledema andthickening are well visualized on CT.􀂄 MRI depicts the para-aortic, aortocavalandmesentric lymph nodes effectively.􀂄GUT TB􀂄 Various patterns of hydronephrosis maybe seenat MR urography.􀂄 MRI helps to differentiate macronodularTBlesions from the other mass lesions.
  • 82. Boehme C, NEJM, 2010
  • 83. CXR Findings Primary TB: Lower or middle lobe infiltrates Reactivated TB: Apical infiltrates/cavitation Latent TB: Usually normal Nodules in hilar area or upper lobes Pleural scarring/thickening
  • 84. Transmission Transmitted by airborne particles 1-5microns in size Ease of transmission depends on durationand proximity of contact as well as thenumber of bacteria excreted Infection can result from only 1-5 bacteriaentering a terminal alveolus Only those with active pulmonary TB areinfectious
  • 85. *M tuberculosis is transmitted via airbornedroplet nuclei that are produced whenpersons with pulmonary or laryngeal TBcough, sneeze, speak, or sing .* Droplet nuclei may be produced by aerosoltreatments, sputum induction,aerosolizationduring bronchoscopy, and throughmanipulation of lesions or processing oftissue or secretions in the hospital orlaboratory.
  • 86. Pathogenesis– Inhalation -> phagocytosis by alveolarmacrophages– Bacterial multiplication occurs intracellularly– Lymphatic spread to regional lymph nodes orhematogenous dissemination– Immune response results in granuloma formation(containment of infection)– Cell death in the granuloma results in caseousnecrosis– Bacteria can remain dormant in the granuloma
  • 87. Pathogenesis– Medical conditions that increase risk foractive TB: Chronic renal failure Diabetes mellitus Silicosis Leukemias/lymphomas Carcinoma of the head/neck or lung Weight loss > 10% of ideal body weight Gastrectomy/jejunoileal bypass
  • 88. Primary pulmonary tuberculosis*The first infection with tubercle bacillus.Includes the involvement of the draininglymph nodes in addition to the initiallesion(Ghon).
  • 89. Clinical features:Majority: symptomless.(specially inyoung adults)Brief febrile illness.Loss of appetite.Failure to gain weight in children.Cough is not unusual and may mimicparoxysm of whooping cough.
  • 90. Physical signs:•May be normal,•Crepitation may be heard.•Primary lesion could beheard.•Segmental or lobar collapsemay occur.
  • 91. Radiological features:•Lymphadenoathy: hilar lymph nodesare most commonly involved rarelyparatracheal.Calciflcation of the nodesmay occur.• Pulmonary componant: ( mainly inadults) segmental or lobarconsolidation or obstructiveemphysema.•Resolution of radiological shadow 6m-2ys.
  • 92. Diagnosis:*Vague ill health with history of contact.* X-ray.*Tuberclin test: is usually stronglypositive.*Sputum and gastric lavage for directsmear and culture helpful in 20-25% ofcases.* DNA amplification: PCR.
  • 93. Post primary pulmonary tuberculosisThe most important type of tuberculosisbecause it is the most frequent andsmear positive sputum is the mainsource of infection responsible for thepersistence of the disease in thecommunity.
  • 94. Source;1. Direct progression of the primarylesion.2. Reactivation of the quiescent primaryor post primary.3. Exogenous infection.
  • 95. Predisposing factors for reactivation:1. Malnutrition.2. Poor housing and overcrowding.3. Steroid and other immunosuppressivedrugs.4. Alcoholism.5.Other diseases: HIV malignancy,lymphomas , Leukaemia,Diabetes.
  • 96. Clinical features:Mainly in middle aged and elderly.A-Symptoms:1. May be no symptoms, or just mild debility.Gradual onset of symptoms over weeks or months.2. General malaise.3. Loss of appetite, loss of weight.4. Febrile course.5. Night sweating.6. Cough with or without sputum.7. Sputum could be mucoid, purulent or blood stained.8. Could be presented with frank haemoptysis.9. Tuberculous pneunonia.
  • 97. B-Signs:1. May be no signs.2. Pallor, cachexia.3. Fever.4. Post tussive crepitations on the apices.5. Signs of Consolidation.6. Signs of fibrosis.7. Signs of cavitary lesion.8. Localised wheezes in endobronchialtuberculosis
  • 98. Lymphnodes Anatomic ConsiderationsRetrosternalPrevascularRetrocavalAortic windowCarinalSubcarinalHilarZ-esophagealCircm-cardiac3 3
  • 99. LymphnodesAnatomic ConsiderationsRetrosternalPrevascularRetrocavalAortic windowCarinalSubcarinalHilarZ-esophagealCircm-cardiac65
  • 100. LymphnodesAnatomic ConsiderationsRetrosternalPrevascularRetrocavalAortic windowCarinalSubcarinalHilarZ-esophagealCircm-cardiac7789
  • 101. Radiology:1. Bilateral upper zone fibrotic shadows: withshift of trachea, mediastinum, distortion offissures and diaphragm, and elevation of thepulmonary hila.2. Soft confluent shadows of exudative lesion(D.D pneumonia)3 Calcification.4. Cavitation.5. Tuberculoma.6. Hilar and paratracheal lymph nodeenlargement may be present.
  • 102. Radiological classification:1.Minimal: slight or moderate opacity. Nocavity. Extent not more than spaceabove 2nd costocondral junction.2. Moderately advanced: In one or bothlungs. slight or moderate opacity, extentequivalent to volume of one lung. Denseconfluent shadow equivalent to one thirdthe volume of one lung. Diameter ofcavities not more than 4 cm.3. Far advanced:Any lesion>the moderately advanced.
  • 103. Diasnosis:1) Clinical2) Plain X-ray.3) Sputum Examination: direct smear and culture (veryimportant).4) Other samples: Gastric aspirate, laryngeal swab, fiberopticspecimens (wash,brush,biopsy),transtracheal spirate.5 Polymerase chain reaction.)6) Tuberclin test: mainly strongly positive7) OthersWhite blood cells if normal favour the diagnosisESR may be elevated.Normocytic normochromic anaemia.CT may be useful in detecting small cavities,or calcification.
  • 104. Miliary TuberculosisProduced by acute dissemination of tuberclebacilli via the blood stream.The term miliaryderives from the radiological picture ofdiffuse, discrete nodular shadows about thesize of millet seed (2mm).
  • 105. A- Classical form:Clinical features:Most common in infants and young children with acuteor subacute febrile illness.In adults: the onset is insidious, gradual vague ill health.Malaise, Cough (usually dry), dyspnea. Night sweat isless common.Headache suggest associated tuberculous meningitisChest examination is free, crepitations may be found.Hepatomegaly, splenomegaly, lymphadenopathy,neck rigidity may be found in rare cases.
  • 106. Diasnosis:1) Clinical.2) Xray.3) Choroidal tubercles in fundus examination4) Tuberclin test not conclusive5) Direct smear and culture of sputum ifpresent.6) Other samples as transtracheal aspirate,fiberoptic specimens may be obtained.7) If failed to prove therapeutic trial for 2weeks
  • 107. Mycobacterium tuberculosis-latent bacilliare microorganisms that adapt to stressfulconditions generated by the infected hostagainst them.By slowing metabolism or becomingdormant, they may counterbalance theseconditions and appear as silent to theimmune system.Moreover, the dynamic turnover of theinfected cells provokes a constantreactivation of the latent bacilli when theenvironmental conditions are favourable, oran activation after being dormant in necroticand fibrotic lesions for a long period of time.
  • 108. Achalasia ofesophagus• Inhomogeneouscardiac density:Right half moredense than left• Density crossingmidline (right blackarrow)• Right sided inlet tooutlet shadow• Right para spinal line(left black arrow)• Barium swallowbelow: Dilatedesophagus
  • 109. Dissecting Aneurysm*Mediastinal widening*Inlet to outlet shadowon left side*Retrocardiac: Intactsilhouette of left heartmargin*Pulmonary arteryoverlay sign: Densitybehind left lower lobe*Wavy margin
  • 110. Treatment Before 1940s: open air (sanatorium) 1946: streptomycin 1952: isoniazid 1970: rifampin
  • 111. Antituberculous drugs:A. First line drugs;Isoniazide (INH) or H Rifampicin ( R ) Pyrizinamide ( Z )Streptomycin ( S ) Ethamutol ( E )B.Second line drugs :Thiacetazone (150mg)Para amino salicylic acid (10-20 g)Ethionamide (<50Kg, 750mg&>50Kg, Ig)Cycloserine 5-20mg/Kg)Kanamycin, Capreomycin, Viomycin (20mg/Kg max Ig)C.New drugs:Amikacin, Quinolones, Rifabutin, new macrolides, andAmoxicillin-clavulinic acid.
  • 112. Drugs Adverse effectDose DoseAdult ChildIsoniazide(INH) or H5 mg/Kg up to12mg/Kg inmiliary.10 mg/Kg Peripheralneuritis,hepatitis,hypersensitivity.Rifampicin(R)lOmg /Kg<50Kg, 450mg>50Kg, 600mg10-20mg Orange urineFlu like illnessHepatitisHypersensitivityBlood dyscriasis.Ethambutol(E)25mg/Kg fortwo months,then 15mg/KgContraindicatedRetrobulbarneuritisPyrazinamie(Z)<50Kg,1.5g50-74Kg, 2g>75Kg, 2.5g40mg/Kg HepatotoxicityHyperuricaemiaStreptomycin(S)20mg/Kg (maxIg)20mg/Kg Ototoxicity(vestibular)NephrotoxicityHypersensitivity
  • 113. Drus regimens:according to WHO guidelines1-New smear positive patient2SRHZ/6HE(8months regimens)or2SHE/10HE(12months regimens)or2SRHZ/4RH (6 months regimen)2-Previously treated smear positive patients2SRHZE/1RHZE/5RHE (8month regimen)a sensitivity pattern is recommended.3- Smear negative and extrapulmonary TB2SHE/10HE (12 months regimen)4- Chronic smear positive patient (Treated inhospital): a Sensitivity pattern is recommended to givespecial treatment regimen.
  • 114. Corticosteroid Therapy in TuberculosisCorticosteroid should never be given to patientswithtuberculosis unless they are receiving adequateantituberculous therapyIndications of steroids:*In very ill patient.*To control drug hypersensitivity.*In tuberculosis of serous sacs (pericarditis, peritonitis andpleural effusion).*In tuberculous meningitis.*Addison disease.*Genitourinary tuberculosis.*Occasionally to suppress lymph node enlargement.
  • 115. Treatment of Active TB Four drug regimen for first 2 months: INH 300 mg Rifampin 600 mg PZA 15-30 mg/kg Ethambutol 15-25 mg/kg or streptomycin 15mg/kg Two drug regimen for next 4 months: INH and rifampin If the TB is not resistant (or < 4%resistance in the community): INH,rifampin, and PZA for the first 2months can be used
  • 116. Treatment of Active TB INH resistant TB:– Rifampin, PZA, and ethambutol for 6months Rifampin resistant TB:– INH, PZA, and streptomycin for 9 monthsor INH and ethambutol for 18 months MDR/XDR TB:– Based on susceptibility patterns
  • 117. Blumberg, HM; IDSA
  • 118. Factors in Treatment ofLatent TB Age: Previously, low risk patients older than 35were not treated – higher risk of drug-induced hepatitis “Decision to tuberculin test is a decision totreat” Weigh risks/benefits of treatment in low riskpatients Liver disease: End stage liver disease and active hepatitisare relative contraindications to treatment
  • 119. Treatment of Latent TB Efficacy of 90% if all the medicationsare taken 60-70% rates when the drugs are self-administered Protective effect will last probably forlife but at least 20 years
  • 120. Treatment of Latent TB Need to exclude active disease beforetreatment (avoids single drug therapyof active TB) CXR – if changes consistent with TB, sendAFB sputum culture Single drug therapy appropriate forlatent TB – bacterial load much lowercompared with active TB
  • 121. Treatment of Latent TB Regimens:– Isoniazid (INH) daily or twice weeklyunder directly observed therapy (esp. ifadherence is an issue) 9 months of treatment is optimal At least 6 months is needed 12 months if treatment is interrupted– Rifampin daily 4 months of treatment Alternative regimen for those exposed to anINH resistant patient
  • 122. Treatment of Latent TB Regimens:– Rifampin/PZA for 2 months Similar in safety and efficacy to 12 monthregimen of INH No longer recommended due to hepatictoxicity (including liver failure leading todeath)
  • 123. Treatment of Latent TB Regimens: Multi-Drug Resistant TB– Therapy based on susceptibility pattern ofthe index case if information available– 2 drug therapy for 12 months PZA plus Ethambutol or Fluoroquinolone with anti-TB activity
  • 124. Treatment of Latent TB If INH treatment is interrupted, anadditional 3 months should be given If interruption is >3 months, re-starttreatment If a treated person is re-exposed tosomeone with TB, repeat treatment isnot needed unless the patient is HIV+
  • 125. Treatment – SpecialSituations HIV: Up to 20% of patients with CD4 counts < 200can have a normal CXR with active TB – sendsputum before treatment Recent contact with someone with active TB:treat regardless of PPD result Anergy testing not recommended Extrapulmonary TB more common Immune reconstitution Same treatment regimens (except rifabutininstead of rifampin for patients on PIs)
  • 126. Treatment – SpecialSituations Pregnancy:– No evidence that PPD testing is harmful– INH: not teratogenic; hepatotoxicity may bemore likely– Rifampin: generally considered safe – reports ofhemorrhage in the newborn– Ethambutol: okay– Streptomycin: avoid (congenital deafness)– PZA: no published safety data– Breast-feeding is not contraindicated
  • 127. Treatment – SpecialSituations Pregnancy:– Active TB – treat– Latent TB (immunocompetent host) –defer therapy until after delivery– Latent TB (HIV or recent converter) –immediate therapy with INH
  • 128. Monitoring on Treatment INH: Side effects Abdominal pain, nausea, vomiting Dark urine Icterus Easy bruising/bleeding Arthralgias Rash Paresthesias/weakness – peripheralneuropathy is less likely with pyridoxine Anorexia/fatigue
  • 129. Monitoring on Treatment INH– Elevated transaminases in 10-20% ofcases – especially with EtOH– Should be withheld if transaminasesincrease more than 3x the upper limit ofnormal when associated with symptomsor 5x the upper limit of normal inasymptomatic patients
  • 130. Monitoring on Treatment Rifampin: Side effects– GI upset– Thrombocytopenia– Hepatitis– Flu-like syndrome – if taken irregularly– Multiple drug interactions– Orange bodily secretions due to excretion
  • 131. Monitoring on Treatment PZA: Side effects– GI upset– Hepatitis– Arthralgias– Hyperuricemia – acute gout uncommon Ethambutol:– Optic neuritis: reversible decreased red-greencolor perception and visual acuity– Not hepatotoxic
  • 132. Adherence Decreases with duration of therapywhereas efficacy increases with lengthof treatment Factors: Side effects Complexity of regimen (active TB) Perception (especially in latent TB) Directly Observed Therapy – especiallyin twice weekly regimens
  • 133. Infection Prevention If active pulmonary TB is suspected:– AFB isolation– Negative pressure– Particulate respirator masks Isolation not required for:– Latent TB– Extrapulmonary TB
  • 134. Infection Prevention Isolation can be discontinued:– If AFB smears x 3 are negative– An alternative diagnosis is made If patient has active TB, then:– After 2 weeks of effective therapy– Resolution of cough, fever– Negative or “less positive” AFB smears
  • 135. S Curve of GoldenWhen there is a mass adjacentto a fissure, the fissure takesthe shape of an "S". Theproximal convexity is due to amass, and the distal concavityis due to atelectasis. Note theshape of the transverse fissure.This example represents a RULmass with atelectasis
  • 136. DefinitionsMDR-TB = Strains resistant to at least INH and RIF (mostimportant 1st-line drugs)XDR-TB = MDR TB strains with additional resistance to anyfluoroquinolone and any of the 3 injectable second-line drugs(amikacin, kanamycin, capreomycin)TDR, XXDR = Resistance to all drugs (not standardised defin)MDR TB XDR TBTB with anydrugresistanceTDR/XXDR TB
  • 137. 2041st-lineoral•INH•RIF•PZA•EMB•(Rfb)Injectables•SM•KM•AMK•CMFluoroquinolones•Cipro•Oflox•Levo•Moxi•(Gati)Oral bacteriostatic2nd lineUnclear efficacy•ETA/PTA•PASA•CYSNot routinely recommended,efficacy unknown, e.g.,amoxacillin/clavulanic acid,clarithromycin, clofazamine,linezolid, inmipenem/cilastatin,high dose isonizidXDR= HR + 1 FQ + 1 Injectable (KM or AMK or CM)