Presentation on theme: "DR.RAGHAVENDRA.H.GOBBUR"— Presentation transcript:
1 DR.RAGHAVENDRA.H.GOBBUR PROFESSOR OF PEDIATRICSB.L.D.E.UNIVERSITY’S Shri.B.M.Patil MEDICAL COLLEGE ,BIJAPUR
2 Newer modalities in TB diagnosis Guest lecture given at state PEDICON 2011DR.RAGHAVENDRA.H.GOBBURPROFESSOR OF PEDIATRICSB.L.D.E.UNIVERSITY’SShri.B.M.Patil MEDICAL COLLEGE ,BIJAPUR.
3 Newer modalities in TB diagnosis IGRA assay Interferon (IFN)-γ AssayMicroscopy LEDCulture :Liquid Medias: BACTEC,BAC T/ALERT 3D, MGITDNA NAAT: Real-time PCR, LIPAEnzyme Assay: ADA
4 QUANTIFERON-TB GOLD in place of Mantoux test . INTERFERON G ASSAY (IGRA)IFN-γ
5 QUANTIFERON-TB GOLD . IN VITRO TEST ESAT-6 ,CFP-10 synthetic peptides are used(Absent in BCG and most NTM)stimulate T-cells from infected peoplereleasing IFN-γ, from These T-cells*early secretory antigenic target-6**culture filtrate protein-10LTBI V/S DISEASE ?TST V/S IGRA
6 QUANTIFERON-TB GOLD . Objective , and controlled test Reduces subjectivity in TB diagnosisSimple diagnostic cut-off (> .35 IU/ml IFN-γ = + )Straight forward positive/negative interpretationEliminates 2 step testingNo ‘booster’ effects in-vitroFaster turn-around, results in hoursResults are electronic (computer generated reports)
7 INDIAN STUDY USING QUANTIFERONTB GOLD Dogra S, Narang P, Mendiratta DK, Chaturvedi P, Reingold AL, Colford JM Jr, Riley LW, Pai M. Comparison of awhole blood interferon-gamma assay with tuberculin skin testing( J Infect 2007; 54:267–76.)Compared QFT to the TST in 105 children ( suspected of TB, or had contact with an index case).11 children (10.5%) were QFT positive, whereas theTST was positive in 15 (15%) at ≥5mm, 11 (10.5%) at ≥10mm, or 4 (4%) at≥15mm.Concordance of TST with QFT was high (95%) at the 10mm TST cut offAll subjects with≥15mm TST , were QFT positive.There wereno indeterminate QFT results, despite 40% children being <4 years old , and 57% of them being malnourished.
8 SUMMARY of IGRA TESTING IGRAs are recommended for 1 SUMMARY of IGRA TESTING IGRAs are recommended for 1. Contacts of active TB Close contacts (HIGH RISK) TST OR IGRA if either is positive, treat for “L TB I” (latent infection) Casual contacts (LOW RISK) can have IGRA confirmation if TST is positive to verify infection v/s BCG or MOTT 2. Immune compromised “suspected child" TST first, if negative do IGRA and if IGRA positive treat as LTBI
9 M.TB. STAINING BY ZEIL-NEILSON STAIN >1,000 Organism per ml sputum required for ordinary microscope.Fluorescent, LED microscope detects even 100 M.TB. organism per ml
11 FIND and Carl Zeiss fluorescent LED microscope based on the proven Primo Star platform.( FIND/Zeiss microscope offers superior optics, reflected light illumination, easy switch from bright field to fluorescent light)11
12 MYCOBACTERIAL CULTURE Culture remains the gold standard for lab confirmation of TBAdvantages:Increases number of case detectionDetects cases among smear negative patientsEstablishes viability of organismsDistinguishing between Mycobacterial speciesHelps in performing DST (drug sensitivity test)Helps in diagnosing cases of treatment failureLimitations:ExpensiveRequire enriched mediaRequire considerable expertiseTime consuming
13 Processing of sputum with CPC Method If delay of more than 48 hours between collection and processing is anticipated, the sputum should be collected with 1%CPC and 2%NaCl2CPC acts as homogenizing and decontaminating agentIt helps in retaining viability of Tubercle bacilli up to daysThese specimens should not be treated with NaOH( Petroff’s)
14 Culture: Extra-Pulmonary Samples Aseptically collected samplesBody fluids:Spinal ,Pleural, Pericardial, Synovial, ascitic, Blood, Pus & Bone marrowTissues:Lymph node, Needle biopsies or Tissue biopsiesSpecimens known to contain contaminating flora:Gastric lavage, Bronchial washings & Urine
17 NEWER CULTURE METHODS for M.TB. Microscopic Observation of Broth Culture &MODS Micro Colony Detection System (slide culture)Septi-check AFB : Non radiometric, Non automatedMGIT : Automated. monitors every 60min O2 utilization, Intensification of O2 quenching fluorescent dyeMB/BAC T - ALERT : Non radiometric, colorimetric detection of CO2BACTEC Radiometric
18 BACTEC 460 TB System(radio metric) Developed in 1969 by Deland and Wagner.PrincipleBACTEC 12B vial , utilize 14C labeled substrate (fatty acid)On inoculation, mycobacteria, grow & release 14CO2.The BACTEC instrument measures quantitatively the radioactivity on a scale ranging from 0-999, as GI.(Growth Indicator)The daily increase in GI is proportional to growth in the medium.DST Drug Susceptibility TestWhen ATT is introduced in the mediumreduced production of 14CO2 and decrease in GI.
20 The MGIT 960 SystemThe MGIT 960 system is a non-radiometric automated system that uses the MGIT media & sensors to detect the fluorescence.Advantages:-The system holds 960 plastic tubes which are continuously monitored.- Early detection with the machine monitoring & reading the tubes every hour.
21 II Mycobacteria Growth Indicator Tube (MGIT) Tube contains modified Middle brook 7H9 broth base with OADC enrichment & PANTA antibiotic mixture.All types of clinical specimens, pulmonary as well as extra-pulmonary ( except blood ) could be cultured on this type of media.
22 The OADC supplement O ----- Oleic acid ( Metabolic stimulant) A Albumin ( to bind toxic free fatty acid )D ---- Dextrose (Energy source )C Catalase ( Destroy toxic peroxides that may be present in the medium )
23 The PANTA antibiotic mixture P Polymyxin BA ---- Amphotericin BN ---- Nalidixic acidT ---- TrimethoprimA Azlocillin+/- VancomycinThe antibiotic mixture inhibits the growth of contaminating bacteria.
24 Principle of the procedure:(MGIT) A fluorescent compound (which is sensitive to O2) is embedded in silicone on the bottom of the tube.The actively respiring microorganisms consume the oxygen & allow the fluorescence to be observed using UV trans-illuminator lamp.
25 III Polymerase Chain Reaction (PCR) & Gene probe Nucleic acid Amplification Tests polymerase enzymes amplify specific DNA sequences, using Nucleic acid probes, using DNA extracted from MTB in the sample.Advantages:- Rapid procedure ( 3 – 4 hours)- High sensitivity (1-10 bacilli / ml sputum)CDC recommends NAAT for all suspected TB cases
26 PCR ASSAYThe thermal cycling, DNA melting separates the strands of DNA double helix at 95°C Heat-stable DNA Taq polymerase, ( Bacteria Thermus aquaticus.) Enzymatically assembles new DNA strands(selectively amplify ) using DNA primers ( DNA oligonucleotides.) & template(each strand) at 55 °C The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
28 REALTIME-PCR ASSAYA TB specific primer and probe mix is provided and this can be detected through the FAM channel.The primer and probe mix exploits the so-called TaqMan® principle.During PCR amplification, forward and reverse primers hybridize to the TB DNA/Cdna. A fluorogenic probe is included in the same reaction mixture , it consists of a DNA probe labelled with a5`-dye (reporter) and a 3`-quencher. (5’3’DQ)During PCR amplification, the probe is cleaved and the Reporter dye and Quencher are separated.The resulting increase in fluorescence can be detected on a range of real-time PCR platforms
29 PCR ASSAYThe PrimerDesign™ genesig Kit for Mycobacterium Tuberculosis (TB) Genomes is designedfor the in vitro quantification of TB genomes.The kit is designed to have the broadest detection profile possible whilst remaining specific to the TB genome.The primers have 100% homology with all other reference sequences in the NCBI database.Fig 1 Accession numbers for detected TB isolates.CP , CP , AM , U , AE , BX , BX ,BX , BX , U , U , AF , BX , BX ,
31 Disadvantages: - Very expensive. - Require specialist training & equipment.- False positive results.( CONTAMINATION)Can not differentiate between living & dead bacilli. .Sputum specimens (3%--7%) might contain inhibitors that prevent or reduce amplification and cause false-negative NAA results.
32 RAPID RECOGNITION OF DRUG RESISTANCE PCR PROBES ARE AVAILABLEKAT-gene INH RESISTANCERPO gene RIFAMPICIN RESISTANCEGYR –A FLUROQUINOLOE RESISTANCELIPA( Line Probe Assay ) amplified DNA is applied to strips with probe for M.TB. And Rif. resistance
33 MODS versus other culture methods* Pos.eachMethod(%)Pos. byatleast one cult.(%)Sens. %MediandetectiondaysAuramine 0769878MODS8997929 (4-31)MGIT88959310 (3-39)LJ739624 ( 6-59)Micro COL 7H117514.5(4-28)PCR8190* Based on 172 samplesCaviedes.L. et al J..Clin.Microbiol. 2000, 38, 1203
34 Identification of M. tuberculosis from the growth Growth temperature 35o-37oC onlyNo pigmentationNiacin positiveCatalase negative at 68oCNo growth on LJ medium containing PNBPositive reaction for nitrate reduction
35 Differentiation of Mycobacteria M.tuberculosisNTMColony morphologyRough, eugonicMostly smoothGrowth at 37oC++/ -Growth at 25oC-PigmentationNiacinPNBNitrate reductionCatalase at 68oC
36 Can high drug dosage still have an effect on resistant strains? IsoniazidMutantskatG – high MICinhA – low MICEarly clinical trialGuinea-pig studyQuinolonesMainly in gyrA – low MIC
37 Evaluation of different methods of diagnosis As regards the time:MGIT shortest time to positivity at 13.3 daysBACTEC 460 system 14.8 days& for L J medium 25.6 days .
38 As regards the no. of culture yield: The best yield, was with BACTEC 460, followed by BACTEC MGIT 960 , & then with L J medium.As regards contamination rate:L J medium (17%) had the highest contamination rate (Tortoli E, Cichero P,Et al. 1999) then the MGIT 960 ( 10.0% )Compared with radiometeric system (3.7%)
39 SUMMARY Useful newer modalities 3idiots? QUANTIFERON-TB GOLDMB BACT-ALERT 3D LIGHT EMITTING SENSORSPCR PROBES for antibiotic resistanceINH,REF,ETH,FLORO Q
44 BACTEC Myco/F�Sputa Culture Medium, for use with the BACTEC 9000MB System to detect mycobacteria species in clinical samples.BACTEC Myco/F Lytic.
45 Dogra S, Narang P, Mendiratta DK, Chaturvedi P, Reingold AL, Colford JM Jr, Riley LW, Pai M. Comparison of a wholeblood interferon-gamma assay with tuberculin skin testingfor the detection of tuberculosis infection in hospitalizedchildren in rural India. J Infect 2007; 54:267–76.An Indian study that compared QFT to the TST in 105 children whowere suspected of having TB, or had contact with an index case.In this study 11 children (10.5%) were QFT positive, whereas the TSTwas positive in 15 (15%) at ≥5mm, 11 (10.5%) at ≥10mm, or 4 (4%) at≥15mm. Concordance of TST with QFT was high (95%) at the 10mmTST cut-off. All ≥15mm TST subjects were QFT positive.There were no indeterminate QFT results, despite 40% of the childrenbeing <4 years old and 57% of them being malnourished.
46 SUMMARY IGRAs are recommended for 1 SUMMARY IGRAs are recommended for 1. Contacts of active TB Close contacts (HIGH RISK) can get both TST and IGRA and if either is positive, be treated for LTBI Casual contacts (LOW RISK) can have IGRA confirmation if TST positive to verify infection vs BCG or MOTT 2. Immune compromised TST first, if negative do IGRA and if IGRA positive treat as LTBI 3. Low risk people who are TST positive Do an IGRA, if positive consider as LTBI
48 FIND and Carl Zeiss Micro Imaging GmbH have co- developed a fluorescent LED microscope based on the proven Primo Star platform. FIND/Zeiss microscope offers superior optics, reflected light illumination, easy switch from brightfield to fluorescent light48
55 INDIAN STUDY USING QUANTIFERONTB GOLD Dogra S, Narang P, Mendiratta DK, Chaturvedi P, Reingold AL, Colford JM Jr, Riley LW, Pai M. Comparison of awhole blood interferon-gamma assay with tuberculin skin testingfor the detection of tuberculosis infection in hospitalizedchildren in rural India. J Infect 2007; 54:267–76.Compared QFT to the TST in 105 children ( suspected of TB, or had contact with an index case).11 children (10.5%) were QFT positive, whereas theTST was positive in 15 (15%) at ≥5mm, 11 (10.5%) at ≥10mm, or 4 (4%) at≥15mm.Concordance of TST with QFT was high (95%) at the 10mmTST cut-off. All ≥15mm TST subjects were QFT positive.There were no indeterminate QFT results, despite 40% of the childrenbeing <4 years old and 57% of them being malnourished.