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Lab diag. tb

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  • NEJM 7/19/01
  • There are two commercial assays available in the world. The first of these, called the QuantiFERON TB test Gold is available in the United States. The test works like this. First blood is drawn into an heparinized tube. The cells are separated and stimulated with various antigens including positive and negative controls. After incubation, sensitized T cells produce interferon that can be harvested and measured using an ELISA assay. The results are interpreted by a computer program.
  • When we measure a person’s TST result, we are measuring and indirect result of inflammation caused by injection of the PPD into the skin. In this figure, you can see that when an antigen presenting cell such as a macrophage encounters a T-cell, the lymphocyte produces a number of cytokines such as TNF-alpha, interferon-gamma, and various interleukins. These cytokines cause swelling and induration that is measured with a ruler. It is now possible to measure these cytokines directly from the blood. Interferon-gamm is the best cytokine to measure because it is produced in large amounts, is stable and thus easy to measure. Importantly it is also a critical cytokine in the immune response to mycobacterial infections.
  • Transcript

    • 1. Hi ! Good Morning ! I am Mycobacterium tuberculosis.
    • 2. LABORATORY DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS
    • 3. NEED FOR ACCURATE & EARLY DIAGNOSIS OF TUBERCULOSIS.
      • Tuberculosis mainly caused by M.tuberculosis.
      • Is a major health problem world wide.
      • Found in Neolithic remains.
      • Largest cause of “DEATHS” from a single infectious disease.
      • HENCE “ACCURATE” & “EARLY” diagnosis is required for its effective “MANAGEMENT”.
    • 4. LABORATORY DIAGNOSIS MAY BE ESTABLISHED BY:
      • Demonstration of bacillus by microscopy.
      • Isolation in culture.
      • Transmitting the infection in experimental animals.
      • Demonstration of hypersensitivity to tuberculoprotein – Mantoux test.
      • Enzyme linked Immunosorbent Spot (ELISPOT) Assay.
    • 5. LABORATORY DIAGNOSIS CONTD.
      • QuantiFeron TB Gold Test.
      • ELISA to detect 38 KDA Mycobacterial Antigen.
      • Molecular methods for early diagnosis.
      • * Amplicor test
      • * E – MTD
      • * DNA sequencing
      • * Line Probe Assay (LiPA)
      • * DNA micro arrays
      • * Molecular beacons.
      • * Single strand conformation Polymorphism (SSCP)
      • * FRET Probes.
      • Other PCR based Techniques.
      • * Amplification Mutation
      • { ARMS }
      • * Branch Migration Inhibition
      • { BMI }
      • Ligase Chain Reaction.
      • Diagnosis of TB & Drug Resistant TB with Mycobacteriophages.
    • 6. SPECIMENS COLLECTED
      • Pulmonary secretions –
      • ** spontaneously produced or induced sputum
      • ** gastric lavage
      • ** transtracheal aspiration
      • ** bronchoscopy
      • ** laryngeal swabbing
      • Gastric lavage specimens.
      • Urine samples.
      • Faecal specimens.
      • Tissue & Body Fluid specimens.
      • Blood specimens
      • Wound ,skin lesion aspirates.
      • Cervical swab.
    • 7. SPUTUM EXAMINED BY :
      • DIRECT METHOD
      • # Z.N.Staining.
      • # Fluorescent Auramine Rhodamine staining
      • AFTER CONCENTRATION
    • 8. Acid-fast bacilli
    • 9. ZN Smear evaluation & AFB Report (Grading of smear) 4+ 01 10 or more 3+ 01 01 – 09 2+ 10 01 – 09 1+ 100 01 – 09 Doubtful; Repeat smear 300 01 – 02 AFB Not seen 300 0 Report No. of OIF No. of AFB
    • 10. Indications for Culture
      • Failures of re-treatment cases
      • Seriously ill cases;
        • extra-pulmonary cases
        • smear negative cases
        • childhood TB & HIV-TB
      • For DRS
      • Not for New Smear Positive Cases
    • 11. Decontamination Procedures
      • 1946 – Trisodium Phosphate
      • 1955 – Pancreatin Desogen
      • 1958 – Pancreatin + 1% cetrimide
      • 1915 – Petroff’s NaOH
      • 1962 – Zephiran Trisodium PO 4
      • 1963 – N-acetyl L- cysteine + 2%NaOH
      • 1969 – Swab culture technique + 1% cetrimide
      • 1975 – CPC + NaCl 2
    • 12. PETROFF’S METHOD
      • Advantages:
      • Simple, inexpensive & control the growth of contaminants
      • Twenty samples can be processed in 2 Hrs, with centrifuge capacity being the limiting factor
      • Sterilized NaOH can be kept for several weeks
      • Limitations:
      • The specimen exposure times must be strictly followed to prevent over kill of tubercle bacilli. The initial kill is independent of additional contributory factors such as heat build-up in the centrifuge and centrifugal efficiency
    • 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%NaCl2
      • CPC acts as homogenizing and decontaminating agent
      • It helps in retaining viability of Tubercle bacilli up to 7 days
      • These specimens should not be treated with NaOH ( Petroff’s)
    • 14. MYCOBACTERIAL CULTURE
      • Advantages :
      • Increases number of cases found
      • Detects cases among smear negative patients
      • Establishes viability of organisms
      • Distinguishing between Mycobacterial species
      • Helps in performing DST
      • Helps in diagnosing cases of failure
      • Limitations :
      • Expensive
      • Require enriched media
      • Require considerable expertise
      • Time consuming
    • 15. Specimen Sterile Non - Sterile Centrifuge & use sediment Liquefaction (N-acetyl-L- cystein) Decontamination NaOH Neutralization Buffer or H2O Centrifugation > 3000 X g Screen by AFB smear & inoculate media (one liquid & one solid) FLOW CHART OF SPECIMEN PROCESSING FOR ISOLATION OF MYCOBACTERIA
    • 16. FLOW CHART CONTD. Screen by AFB smear & inoculate media (one liquid & one solid) Liquid Medium Solid Media MGIT BACTEC SEPTI-CHEK CMS Incubate At 37 ºC For 6 wks Incubate At 37ºC For 6 wks Incubate inverting At 37ºC For 8 wks Incubate At 37ºC For 6 wks Fluoresc- -ence detected Growth Index >10 Colonies or turbidity Growth detected Confirm by AFB smear Reinoculate on Solid media L J L J with RNA LJ with Pyruvic acid Incubate At 37ºC For 8 wks If growth Confirm on AFB smear
    • 17. Reading and Reporting
      • Characteristics of Tubercle bacilli
      • Growth of Primary culture takes 2 – 4 weeks to obtain visible colonies
      • Colonies are buff colored and rough, having the appearance of bread crumbs or cauliflower
      • Not easily emulsified but give a granular suspension
      • Microscopically frequently arranged in serpentine cords of varying length or show linear clumping
    • 18. Culture Media : Solid
      • LJ
      • LJ with Na pyruvate
      • LJ with out asparagine
      • Middlebrook’s 7H10 & 7H11
      • Selective 7H10 & 11
      • Ogawa
      • Tarshi’s Blood Agar
    • 19. Slow growth 3-4 wk
    • 20. 24-hr generation time AFB AFB AFB 24 hr
    • 21. MYCOBACTERIAL COLONIES ON SOLID MEDIA
    • 22. Colonies growing on media
    • 23. Laboratory diagnosis
      • Traditional (slow)
        • Growth on solid media
        • Biochemical tests to speciate
      • Recent
        • Radiometric growth detection
        • BACTEC
        • SEPTI – CHEK
        • MGIT
        • Gene probes to speciate
    • 24. Flow Chart Identification of Tubercle bacilli & related Growth on LJ Medium Rapid growth within 7 days Growth on MacConkey Agar Aryl-sulphataes test Slow Growth Niacin test
      • Ve
      • Tellurite Reduction
      + ve M. smegmatis
      • ve
      • M. phlei
      - ve Type of growth + ve M. tuberculosis + ve M. Fortuitum complex Pigment Scanty Smooth Flat Colonies In Light Group I Photochromogens In Dark Group II Scotochromogens No Pigment Group III Non - Chromogens - ve BCG + ve M. bovis M.Kansasi M.intermedium M.Szulgai M.scrofulaceum M.Avium complex M.gastri
    • 25. Other Culture Methods
      • Septi-check AFB
      • MGIT 960
      • Backtec/MB/Bact
      • ESP Culture ii
      • M icroscopic O bservation of B roth C ulture
      • MODS: M icro C olony D etection S ystem
    • 26. BACTEC CONTINUOUS MONITORING SYSTEM
    • 27. BLOOD CULTURE BOTTLES FOR BACTEC 9240,9120,9050.
    • 28. SEPTI - CHECK
    • 29. Radiolabeled palmitic acid AFB *C___ *C___ *C___ *C___
    • 30. Detect growth with CO 2 AFB AFB AFB AFB AFB *CO 2 *CO 2 *C___ *C___
    • 31. BIOCHEMICAL REACTIONS: + - +/- - - - - - - - M Africanum + - - +/- - - - - - - M bovis + + + +/- - + - + - + M tuberculosis UREASE TEST PYRAZI-NAMIDASE TSET GROWTH ON TCH TELLURI--TE REDUCTION TEST TWEEN 80 HYDRO--LYSIS TSET PEROX---IDASE TEST HOT CATAL---ASE TEST NITRATE REDUC---TION TEST ARYL-SULPH---ATASE TEST NIACIN TEST SPECIES
    • 32. BIOCHEMICAL REACTIONS
    • 33. BIOCHEMICAL REACTIONS
    • 34. BIOCHEMICAL REACTIONS
    • 35. BIOCHEMICAL REACTIONS
    • 36. ANIMAL INOCULATION
      • Intramuscular injection of the bacterial
      • concentrated material into two healthy
      • guinea pigs of 12 week and autopsied,
      • one after four weeks & second after eight
      • weeks of inoculation.
    • 37. ANTIGEN PROTEIN DETECTION
      • Tuberculostearic acid : a fatty extracted from
      • the cell wall of M.tuberculosis detected by gas
      • chromatography/ mass spectrometry in clinical
      • samples. M.tuberculosis is unique to release
      • tuberculostearic acid .
      • Host enzyme Adenosine deaminase : Host
      • enzyme. It is increased in infection caused by
      • M.tuberculosis.
    • 38. CHROMATOGRAPHIC ANALYSIS
      • Analysis of mycobacterial lipids by ----
      • * Thin paper chromatography
      • * Gas liquid chromatography (GLC)
      • * Reverse phase high performance liquid chromatography ( HPLC)
      • HPLC of extracted mycobacteria -
      • * A very rapid & specific method for identification of species.
    • 39. Why Measure Interferon-  ?
      • TB infection induces T-cell response (CMI)
      • IFN-  is the ‘classic’ CMI cytokine
      • Produced in vitro in response to specific antigen
      • Secreted in measurable and stable amounts
      • Absent from normal circulation
      • Extensive literature showing importance of IFN-  in TB infection
    • 40. What is Quanti-FERON ® -TB Gold
      • Blood assay for M. tuberculosis > Interferon γ release assay
      • In vitro test using whole blood specimen for the diagnosis of TB infection, whether latent or active
      • Does not distinguish between latent TB infection or TB disease
    • 41. Quanti-FERON ® -TB Gold – Scientific Basis
      • This recognition process involves the generation of interferon- γ , a specific cytokine for cell mediated immune response
      • Individuals infected with M. tuberculosis complex organisms have lymphocytes in their blood that recognize mycobacterial antigens
      • The detection and subsequent quantification of IFN- γ is the basis of this test
      • The test uses synthetic peptide antigens (ESAT-6, CFP-10) that simulate mycobacterial proteins to generate the immune response
    • 42. Interferon Gamma Release
    • 43. Whole Blood IFN-  Assay QuantiFERON-TB Test Cellestis ESAT-6 CFP 10 Mitogen Control TMB COLOR Stage 1 Whole Blood Culture Stage 2 IFN-gamma ELISA Nil Control Incubate -> INF-  from sensitized T-cells Draw blood + heparin Aliquot blood & add antigen Harvest plasma from above settled cells Measure [ IFN-  ] in ‘Sandwich’ ELISA Computerized interpretation
    • 44. Species Specificity of ESAT-6 and CFP-10
    • 45. QFT Assay
    • 46. In Vivo and In Vitro Diagnostic Tests Antigen presenting cell Memory T-cell Presentation of mycobacterial antigens IFN-  IFN-  IL-8, etc. IL-8, etc. TNF-  TNF- 
    • 47. Results and Interpretation MTB infection status cannot be determined as a result of impaired immunity and/or incorrect performance of the test INDETERMINATE No ESAT-6 or CFP-10 responsiveness detected M. tuberculosis unlikely NEGATIVE ESAT-6 and/or CFP-10 responsiveness detected M. tuberculosis infection likely POSITIVE INTERPRETATION RESULT
    • 48. QFT and TST
      • QFT
      • in vitro test
      • Specific antigens
      • No boosting
      • 1 patient visit
      • Lab variability
      • Results possible in 1 day
      • Requires phlebotomy
      • Includes + control
      • TST
      • in vivo test
      • Less specific PPD
      • Boosting
      • 2 patient visits
      • Inter-reader variability
      • Results in 2-3 days
      • No phlebotomy required
      • No + control
    • 49. T-Spot. TB  : “Six easy Steps” Oxford Immunotec Nil Control Positive Control Infection Infection
    • 50. ENZYME LINKED IMMUNOSORBENT SPOT (ELISPOT) TEST.
      • It is based on ELISA test
      • Allows visualization of secretory products of individual activated cells.
      • Provides information about type of immune protein (qualitative)& number of responding cells (quantitative)
    • 51. USING ELISA TO DETECT 38kDA MYCOBACTERIAL ANTIGEN
      • 38 kDA secretory protein being one of the
      • most important specific antigens of MTB
      • It induses B & T cell responses with high
      • specificity to MTB
      • Detected by Direct & Sandwich ELISA.
    • 52. Mantoux Tuberculin Skin Test
      • Preferred method of testing for TB infection in adults and children
      • Tuberculin skin testing useful for
        • Examining person who is not ill but may be infected
        • Determining how many people in group are infected
        • Examining person who has symptoms of TB
    • 53. Administering the Tuberculin Skin Test
      • Inject intradermally 0.1 ml of 5
      • TU PPD tuberculin
      • Produce wheal 6 mm to 10 mm
      • in diameter
      • Do not recap, bend, or break
      • needles, or remove needles from syringes
      • Follow universal precautions for infection control
    • 54. Reading the Tuberculin Skin Test
      • Read reaction 48-72
      • Hours after injection
      • Measure only induration
      • Record reaction in
      • millimeters
    • 55. Classifying the Tuberculin Reaction
      • > 5 mm is classified as positive in
      • HIV-positive persons
      • Recent contacts of TB case
      • Persons with fibrotic changes on chest radiograph consistent with old healed TB
      • Patients with organ transplants and other
      • immunosuppressed patients
    • 56. Classifying the Tuberculin Reaction (cont.)
      • > 10 mm is classified as positive in
      • Recent arrivals from high-prevalence countries
      • Injection drug users
      • Residents and employees of high-risk congregate settings
      • Mycobacteriology laboratory personnel
      • Persons with clinical conditions that place them at high risk
      • Children <4 years of age, or children and adolescents
      • exposed to adults in high-risk categories
    • 57. Classifying the Tuberculin Reaction (cont.)
      • > 15 mm is classified as positive in
      • Persons with no known risk factors for TB
      • Targeted skin testing programs should only be conducted among high-risk groups
    • 58. BCG and TST (1)
      • General teaching is that reactivity from BCG wanes after a few years and is unlikely to persist > 10 years, but may be boosted by PPD.
      • Study done in Switzerland* suggests that false positives due to BCG may be much more common than we thought:
        • 40% of 5000 HCW had positive TST
        • Prior BCG strongest risk factor for positive TST among those less than age 40 with TSTs < 18 mm (was not as strong a risk factor for those > 40 years old and those with TSTs > 20 mm)
    • 59. BCG and TST (2)
      • Review of studies that compared TST responses to BCG during and after infancy
      • Vaccination during infancy estimated to cause false-positive TST in 6.3% overall, but only 1% of those tested more than 10 years after vaccination
      • Vaccination at 2 years of age or older estimated to cause false-positive TST in 40% of persons overall, 20% of those tested 10 years or more after vaccination
      Farhat M et al, Int J Tuberc Lung Dis 2006; 10: 1192-204
    • 60. Nucleic acid amplification for mycobact. diagnosis Genus specific protocols Targeting genes code for 16S rRNA 65KDa hsp M.TB Complex specific is 6110 Other targets: Genes encoding 38 KDa MPB 64 mtp 40 PMT 64 Methods: Target amplification - PCR (TMA, LCR, SDA or signal amplification EG: QB amplification) PFYFFER G.E. J.INF. 1999, 39 , 21-26. TC/ICM 30
    • 61. MOLECULAR TECHNIQUES TO DETECT M.TUBERCULOSIS - 1
      • Amplicor Test
      • ** Detects the presence of the
      • mycobacterial 16S ribosomal ( rRNA)
      • gene by the PCR amplification
      • followed by ELISA reaction.
    • 62. Mycobacteria (AFB) sample U AFB U
    • 63. Extract 16S RNA U U 16S RNA
    • 64. Probe for specific 16S RNA U U 16S RNA Probe Probe
    • 65. Probe remains in sample U U 16S RNA Probe
    • 66. Detector binds to probe ******* U ******* U 16S RNA Probe
    • 67. Detector remains in sample ******* U U 16S RNA Probe
    • 68. Detector identifies 16S RNA ******* VVVVV U U 16S RNA Probe
    • 69. MOLECULAR TECHNIQUES TO DETECT M.TUBERCULOSIS - 2
      • E – MTD
      • ** The assay is based on the transcription mediated amplification system ( TMA ).The mycobacterial rRNA from target cells is released by sonication & amplified a billion fold by TMA.
    • 70. Conventional method for diagnosis of drug resistant M.tuberculosis
      • Biochemical tests : Catalase & Peroxidase tests
      • *** BOTH ARE NEGATIVE IN INH RESISTANT M.TUBERCULOSIS.
    • 71. SENSITIVITY TESTS
      • Absolute conc. Method : No.of media containing serial conc. Of the drugs are inoculated & minimum inhibitory conc.calculated.
      • Resistance Ratio Method : Two sets of media containing graded conc.of drugs inoculated. One with test strain & other with standard strain of known sensitivity.
      • Proportion Method : Average sensitivity of the strain.
    • 72. Various Mycobacterial gene mutations involved in drug resistance.
      • rpoB gene mutation --- Rifampicin
      • katG & inhA mutation --- INH
      • rpsL gene mutation --- Streptomycin
      • pncA gene mutation --- Pyrazinamide
      • embB gene mutation --- Ethambutol
    • 73. Molecular diagnostic methods for drug resistant M.tuberculosis
      • DNA Sequencing : reliable & accurate.
      • Line Probe Assay (LiPA) : to identify mutations in the rpoB core gene.
      • DNA microarrays : based on principle of hybridization
      • Molecular beacons : they are hair-pin shaped probes to detect presence of specific nucleic acids.
      • Single strand conformation polymorphism : determines presence of mutations in specific DNA regions
      • FRET ( Fluorescent resonance energy transfer) probes: used to detect presence of mutations in real time PCR.
    • 74. Other PCR based tests:
      • Amplification Refractory Mutation System: has been applied to know mutations in rpoB, katG, embB, genes.
      • Branch Migration Inhibition : spontaneous strand exchange is inhibited by sequence difference between two DNA molecules.
    • 75. Diagnosis of drug resistance with Mycobacteriophages
      • Phage amplified biological assay (phaB)
      • Luciferase reporter phages (LRPs)
    • 76. Antituberculosis Drugs Currently in Use
      • First-line Drugs
        • Isoniazid
        • Rifampin
        • Rifapentine
        • Rifabutin
        • Ethambutol
        • Pyrazinamide
      • Second-line Drugs
        • Cycloserine
        • Ethionamide
        • Levofloxacin
        • Moxifloxacin
        • Gatifloxacin
        • P -Aminosalicylic acid
        • Streptomycin
        • Amikacin/kanamycin
        • Capreomycin
        • Linezolid
    • 77.
      • -BCG
      PREVENTION CHALLENGES WHO Jan 07 “ BCG vaccine should not be used in children known to be HIV-infected”
    • 78. THANK YOU