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Lab diag. tb


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Lab diag. tb

  1. 1. Hi ! Good Morning ! I am Mycobacterium tuberculosis.
  3. 3. NEED FOR ACCURATE & EARLY DIAGNOSIS OF TUBERCULOSIS. <ul><li>Tuberculosis mainly caused by M.tuberculosis. </li></ul><ul><li>Is a major health problem world wide. </li></ul><ul><li>Found in Neolithic remains. </li></ul><ul><li>Largest cause of “DEATHS” from a single infectious disease. </li></ul><ul><li>HENCE “ACCURATE” & “EARLY” diagnosis is required for its effective “MANAGEMENT”. </li></ul>
  4. 4. LABORATORY DIAGNOSIS MAY BE ESTABLISHED BY: <ul><li>Demonstration of bacillus by microscopy. </li></ul><ul><li>Isolation in culture. </li></ul><ul><li>Transmitting the infection in experimental animals. </li></ul><ul><li>Demonstration of hypersensitivity to tuberculoprotein – Mantoux test. </li></ul><ul><li>Enzyme linked Immunosorbent Spot (ELISPOT) Assay. </li></ul>
  5. 5. LABORATORY DIAGNOSIS CONTD. <ul><li>QuantiFeron TB Gold Test. </li></ul><ul><li>ELISA to detect 38 KDA Mycobacterial Antigen. </li></ul><ul><li>Molecular methods for early diagnosis. </li></ul><ul><li>* Amplicor test </li></ul><ul><li>* E – MTD </li></ul><ul><li>* DNA sequencing </li></ul><ul><li>* Line Probe Assay (LiPA) </li></ul><ul><li>* DNA micro arrays </li></ul><ul><li>* Molecular beacons. </li></ul><ul><li>* Single strand conformation Polymorphism (SSCP) </li></ul><ul><li>* FRET Probes. </li></ul><ul><li>Other PCR based Techniques. </li></ul><ul><li>* Amplification Mutation </li></ul><ul><li>{ ARMS } </li></ul><ul><li>* Branch Migration Inhibition </li></ul><ul><li>{ BMI } </li></ul><ul><li>Ligase Chain Reaction. </li></ul><ul><li>Diagnosis of TB & Drug Resistant TB with Mycobacteriophages. </li></ul>
  6. 6. SPECIMENS COLLECTED <ul><li>Pulmonary secretions – </li></ul><ul><li>** spontaneously produced or induced sputum </li></ul><ul><li>** gastric lavage </li></ul><ul><li>** transtracheal aspiration </li></ul><ul><li>** bronchoscopy </li></ul><ul><li>** laryngeal swabbing </li></ul><ul><li>Gastric lavage specimens. </li></ul><ul><li>Urine samples. </li></ul><ul><li>Faecal specimens. </li></ul><ul><li>Tissue & Body Fluid specimens. </li></ul><ul><li>Blood specimens </li></ul><ul><li>Wound ,skin lesion aspirates. </li></ul><ul><li>Cervical swab. </li></ul>
  7. 7. SPUTUM EXAMINED BY : <ul><li>DIRECT METHOD </li></ul><ul><li># Z.N.Staining. </li></ul><ul><li># Fluorescent Auramine Rhodamine staining </li></ul><ul><li>AFTER CONCENTRATION </li></ul>
  8. 8. Acid-fast bacilli
  9. 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. 10. Indications for Culture <ul><li>Failures of re-treatment cases </li></ul><ul><li>Seriously ill cases; </li></ul><ul><ul><li>extra-pulmonary cases </li></ul></ul><ul><ul><li>smear negative cases </li></ul></ul><ul><ul><li>childhood TB & HIV-TB </li></ul></ul><ul><li>For DRS </li></ul><ul><li>Not for New Smear Positive Cases </li></ul>
  11. 11. Decontamination Procedures <ul><li>1946 – Trisodium Phosphate </li></ul><ul><li>1955 – Pancreatin Desogen </li></ul><ul><li>1958 – Pancreatin + 1% cetrimide </li></ul><ul><li>1915 – Petroff’s NaOH </li></ul><ul><li>1962 – Zephiran Trisodium PO 4 </li></ul><ul><li>1963 – N-acetyl L- cysteine + 2%NaOH </li></ul><ul><li>1969 – Swab culture technique + 1% cetrimide </li></ul><ul><li>1975 – CPC + NaCl 2 </li></ul>
  12. 12. PETROFF’S METHOD <ul><li>Advantages: </li></ul><ul><li>Simple, inexpensive & control the growth of contaminants </li></ul><ul><li>Twenty samples can be processed in 2 Hrs, with centrifuge capacity being the limiting factor </li></ul><ul><li>Sterilized NaOH can be kept for several weeks </li></ul><ul><li>Limitations: </li></ul><ul><li>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 </li></ul>
  13. 13. Processing of sputum with CPC Method <ul><li>If delay of more than 48 hours between collection and processing is anticipated, the sputum should be collected with 1%CPC and 2%NaCl2 </li></ul><ul><li>CPC acts as homogenizing and decontaminating agent </li></ul><ul><li>It helps in retaining viability of Tubercle bacilli up to 7 days </li></ul><ul><li>These specimens should not be treated with NaOH ( Petroff’s) </li></ul>
  14. 14. MYCOBACTERIAL CULTURE <ul><li>Advantages : </li></ul><ul><li>Increases number of cases found </li></ul><ul><li>Detects cases among smear negative patients </li></ul><ul><li>Establishes viability of organisms </li></ul><ul><li>Distinguishing between Mycobacterial species </li></ul><ul><li>Helps in performing DST </li></ul><ul><li>Helps in diagnosing cases of failure </li></ul><ul><li>Limitations : </li></ul><ul><li>Expensive </li></ul><ul><li>Require enriched media </li></ul><ul><li>Require considerable expertise </li></ul><ul><li>Time consuming </li></ul>
  15. 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. 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. 17. Reading and Reporting <ul><li>Characteristics of Tubercle bacilli </li></ul><ul><li>Growth of Primary culture takes 2 – 4 weeks to obtain visible colonies </li></ul><ul><li>Colonies are buff colored and rough, having the appearance of bread crumbs or cauliflower </li></ul><ul><li>Not easily emulsified but give a granular suspension </li></ul><ul><li>Microscopically frequently arranged in serpentine cords of varying length or show linear clumping </li></ul>
  18. 18. Culture Media : Solid <ul><li>LJ </li></ul><ul><li>LJ with Na pyruvate </li></ul><ul><li>LJ with out asparagine </li></ul><ul><li>Middlebrook’s 7H10 & 7H11 </li></ul><ul><li>Selective 7H10 & 11 </li></ul><ul><li>Ogawa </li></ul><ul><li>Tarshi’s Blood Agar </li></ul>
  19. 19. Slow growth 3-4 wk
  20. 20. 24-hr generation time AFB AFB AFB 24 hr
  22. 22. Colonies growing on media
  23. 23. Laboratory diagnosis <ul><li>Traditional (slow) </li></ul><ul><ul><li>Growth on solid media </li></ul></ul><ul><ul><li>Biochemical tests to speciate </li></ul></ul><ul><li>Recent </li></ul><ul><ul><li>Radiometric growth detection </li></ul></ul><ul><ul><li>BACTEC </li></ul></ul><ul><ul><li>SEPTI – CHEK </li></ul></ul><ul><ul><li>MGIT </li></ul></ul><ul><ul><li>Gene probes to speciate </li></ul></ul>
  24. 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 <ul><li>Ve </li></ul><ul><li>Tellurite Reduction </li></ul>+ ve M. smegmatis <ul><li>ve </li></ul><ul><li>M. phlei </li></ul>- 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. 25. Other Culture Methods <ul><li>Septi-check AFB </li></ul><ul><li>MGIT 960 </li></ul><ul><li>Backtec/MB/Bact </li></ul><ul><li>ESP Culture ii </li></ul><ul><li>M icroscopic O bservation of B roth C ulture </li></ul><ul><li>MODS: M icro C olony D etection S ystem </li></ul>
  27. 27. BLOOD CULTURE BOTTLES FOR BACTEC 9240,9120,9050.
  28. 28. SEPTI - CHECK
  29. 29. Radiolabeled palmitic acid AFB *C___ *C___ *C___ *C___
  30. 30. Detect growth with CO 2 AFB AFB AFB AFB AFB *CO 2 *CO 2 *C___ *C___
  36. 36. ANIMAL INOCULATION <ul><li>Intramuscular injection of the bacterial </li></ul><ul><li>concentrated material into two healthy </li></ul><ul><li>guinea pigs of 12 week and autopsied, </li></ul><ul><li>one after four weeks & second after eight </li></ul><ul><li>weeks of inoculation. </li></ul>
  37. 37. ANTIGEN PROTEIN DETECTION <ul><li>Tuberculostearic acid : a fatty extracted from </li></ul><ul><li>the cell wall of M.tuberculosis detected by gas </li></ul><ul><li>chromatography/ mass spectrometry in clinical </li></ul><ul><li>samples. M.tuberculosis is unique to release </li></ul><ul><li>tuberculostearic acid . </li></ul><ul><li>Host enzyme Adenosine deaminase : Host </li></ul><ul><li>enzyme. It is increased in infection caused by </li></ul><ul><li>M.tuberculosis. </li></ul>
  38. 38. CHROMATOGRAPHIC ANALYSIS <ul><li>Analysis of mycobacterial lipids by ---- </li></ul><ul><li>* Thin paper chromatography </li></ul><ul><li>* Gas liquid chromatography (GLC) </li></ul><ul><li>* Reverse phase high performance liquid chromatography ( HPLC) </li></ul><ul><li>HPLC of extracted mycobacteria - </li></ul><ul><li>* A very rapid & specific method for identification of species. </li></ul>
  39. 39. Why Measure Interferon-  ? <ul><li>TB infection induces T-cell response (CMI) </li></ul><ul><li>IFN-  is the ‘classic’ CMI cytokine </li></ul><ul><li>Produced in vitro in response to specific antigen </li></ul><ul><li>Secreted in measurable and stable amounts </li></ul><ul><li>Absent from normal circulation </li></ul><ul><li>Extensive literature showing importance of IFN-  in TB infection </li></ul>
  40. 40. What is Quanti-FERON ® -TB Gold <ul><li>Blood assay for M. tuberculosis > Interferon γ release assay </li></ul><ul><li>In vitro test using whole blood specimen for the diagnosis of TB infection, whether latent or active </li></ul><ul><li>Does not distinguish between latent TB infection or TB disease </li></ul>
  41. 41. Quanti-FERON ® -TB Gold – Scientific Basis <ul><li>This recognition process involves the generation of interferon- γ , a specific cytokine for cell mediated immune response </li></ul><ul><li>Individuals infected with M. tuberculosis complex organisms have lymphocytes in their blood that recognize mycobacterial antigens </li></ul><ul><li>The detection and subsequent quantification of IFN- γ is the basis of this test </li></ul><ul><li>The test uses synthetic peptide antigens (ESAT-6, CFP-10) that simulate mycobacterial proteins to generate the immune response </li></ul>
  42. 42. Interferon Gamma Release
  43. 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. 44. Species Specificity of ESAT-6 and CFP-10
  45. 45. QFT Assay
  46. 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. 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. 48. QFT and TST <ul><li>QFT </li></ul><ul><li>in vitro test </li></ul><ul><li>Specific antigens </li></ul><ul><li>No boosting </li></ul><ul><li>1 patient visit </li></ul><ul><li>Lab variability </li></ul><ul><li>Results possible in 1 day </li></ul><ul><li>Requires phlebotomy </li></ul><ul><li>Includes + control </li></ul><ul><li>TST </li></ul><ul><li>in vivo test </li></ul><ul><li>Less specific PPD </li></ul><ul><li>Boosting </li></ul><ul><li>2 patient visits </li></ul><ul><li>Inter-reader variability </li></ul><ul><li>Results in 2-3 days </li></ul><ul><li>No phlebotomy required </li></ul><ul><li>No + control </li></ul>
  49. 49. T-Spot. TB  : “Six easy Steps” Oxford Immunotec Nil Control Positive Control Infection Infection
  50. 50. ENZYME LINKED IMMUNOSORBENT SPOT (ELISPOT) TEST. <ul><li>It is based on ELISA test </li></ul><ul><li>Allows visualization of secretory products of individual activated cells. </li></ul><ul><li>Provides information about type of immune protein (qualitative)& number of responding cells (quantitative) </li></ul>
  51. 51. USING ELISA TO DETECT 38kDA MYCOBACTERIAL ANTIGEN <ul><li>38 kDA secretory protein being one of the </li></ul><ul><li>most important specific antigens of MTB </li></ul><ul><li>It induses B & T cell responses with high </li></ul><ul><li>specificity to MTB </li></ul><ul><li>Detected by Direct & Sandwich ELISA. </li></ul>
  52. 52. Mantoux Tuberculin Skin Test <ul><li>Preferred method of testing for TB infection in adults and children </li></ul><ul><li>Tuberculin skin testing useful for </li></ul><ul><ul><li>Examining person who is not ill but may be infected </li></ul></ul><ul><ul><li>Determining how many people in group are infected </li></ul></ul><ul><ul><li>Examining person who has symptoms of TB </li></ul></ul>
  53. 53. Administering the Tuberculin Skin Test <ul><li>Inject intradermally 0.1 ml of 5 </li></ul><ul><li>TU PPD tuberculin </li></ul><ul><li>Produce wheal 6 mm to 10 mm </li></ul><ul><li>in diameter </li></ul><ul><li>Do not recap, bend, or break </li></ul><ul><li>needles, or remove needles from syringes </li></ul><ul><li>Follow universal precautions for infection control </li></ul>
  54. 54. Reading the Tuberculin Skin Test <ul><li>Read reaction 48-72 </li></ul><ul><li>Hours after injection </li></ul><ul><li>Measure only induration </li></ul><ul><li>Record reaction in </li></ul><ul><li>millimeters </li></ul>
  55. 55. Classifying the Tuberculin Reaction <ul><li>> 5 mm is classified as positive in </li></ul><ul><li>HIV-positive persons </li></ul><ul><li>Recent contacts of TB case </li></ul><ul><li>Persons with fibrotic changes on chest radiograph consistent with old healed TB </li></ul><ul><li>Patients with organ transplants and other </li></ul><ul><li>immunosuppressed patients </li></ul>
  56. 56. Classifying the Tuberculin Reaction (cont.) <ul><li>> 10 mm is classified as positive in </li></ul><ul><li>Recent arrivals from high-prevalence countries </li></ul><ul><li>Injection drug users </li></ul><ul><li>Residents and employees of high-risk congregate settings </li></ul><ul><li>Mycobacteriology laboratory personnel </li></ul><ul><li>Persons with clinical conditions that place them at high risk </li></ul><ul><li>Children <4 years of age, or children and adolescents </li></ul><ul><li>exposed to adults in high-risk categories </li></ul>
  57. 57. Classifying the Tuberculin Reaction (cont.) <ul><li>> 15 mm is classified as positive in </li></ul><ul><li>Persons with no known risk factors for TB </li></ul><ul><li>Targeted skin testing programs should only be conducted among high-risk groups </li></ul>
  58. 58. BCG and TST (1) <ul><li>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. </li></ul><ul><li>Study done in Switzerland* suggests that false positives due to BCG may be much more common than we thought: </li></ul><ul><ul><li>40% of 5000 HCW had positive TST </li></ul></ul><ul><ul><li>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) </li></ul></ul>
  59. 59. BCG and TST (2) <ul><li>Review of studies that compared TST responses to BCG during and after infancy </li></ul><ul><li>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 </li></ul><ul><li>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 </li></ul>Farhat M et al, Int J Tuberc Lung Dis 2006; 10: 1192-204
  60. 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. 61. MOLECULAR TECHNIQUES TO DETECT M.TUBERCULOSIS - 1 <ul><li>Amplicor Test </li></ul><ul><li>** Detects the presence of the </li></ul><ul><li>mycobacterial 16S ribosomal ( rRNA) </li></ul><ul><li>gene by the PCR amplification </li></ul><ul><li>followed by ELISA reaction. </li></ul>
  62. 62. Mycobacteria (AFB) sample U AFB U
  63. 63. Extract 16S RNA U U 16S RNA
  64. 64. Probe for specific 16S RNA U U 16S RNA Probe Probe
  65. 65. Probe remains in sample U U 16S RNA Probe
  66. 66. Detector binds to probe ******* U ******* U 16S RNA Probe
  67. 67. Detector remains in sample ******* U U 16S RNA Probe
  68. 68. Detector identifies 16S RNA ******* VVVVV U U 16S RNA Probe
  69. 69. MOLECULAR TECHNIQUES TO DETECT M.TUBERCULOSIS - 2 <ul><li>E – MTD </li></ul><ul><li>** 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. </li></ul>
  70. 70. Conventional method for diagnosis of drug resistant M.tuberculosis <ul><li>Biochemical tests : Catalase & Peroxidase tests </li></ul><ul><li>*** BOTH ARE NEGATIVE IN INH RESISTANT M.TUBERCULOSIS. </li></ul>
  71. 71. SENSITIVITY TESTS <ul><li>Absolute conc. Method : No.of media containing serial conc. Of the drugs are inoculated & minimum inhibitory conc.calculated. </li></ul><ul><li>Resistance Ratio Method : Two sets of media containing graded conc.of drugs inoculated. One with test strain & other with standard strain of known sensitivity. </li></ul><ul><li>Proportion Method : Average sensitivity of the strain. </li></ul>
  72. 72. Various Mycobacterial gene mutations involved in drug resistance. <ul><li>rpoB gene mutation --- Rifampicin </li></ul><ul><li>katG & inhA mutation --- INH </li></ul><ul><li>rpsL gene mutation --- Streptomycin </li></ul><ul><li>pncA gene mutation --- Pyrazinamide </li></ul><ul><li>embB gene mutation --- Ethambutol </li></ul>
  73. 73. Molecular diagnostic methods for drug resistant M.tuberculosis <ul><li>DNA Sequencing : reliable & accurate. </li></ul><ul><li>Line Probe Assay (LiPA) : to identify mutations in the rpoB core gene. </li></ul><ul><li>DNA microarrays : based on principle of hybridization </li></ul><ul><li>Molecular beacons : they are hair-pin shaped probes to detect presence of specific nucleic acids. </li></ul><ul><li>Single strand conformation polymorphism : determines presence of mutations in specific DNA regions </li></ul><ul><li>FRET ( Fluorescent resonance energy transfer) probes: used to detect presence of mutations in real time PCR. </li></ul>
  74. 74. Other PCR based tests: <ul><li>Amplification Refractory Mutation System: has been applied to know mutations in rpoB, katG, embB, genes. </li></ul><ul><li>Branch Migration Inhibition : spontaneous strand exchange is inhibited by sequence difference between two DNA molecules. </li></ul>
  75. 75. Diagnosis of drug resistance with Mycobacteriophages <ul><li>Phage amplified biological assay (phaB) </li></ul><ul><li>Luciferase reporter phages (LRPs) </li></ul>
  76. 76. Antituberculosis Drugs Currently in Use <ul><li>First-line Drugs </li></ul><ul><ul><li>Isoniazid </li></ul></ul><ul><ul><li>Rifampin </li></ul></ul><ul><ul><li>Rifapentine </li></ul></ul><ul><ul><li>Rifabutin </li></ul></ul><ul><ul><li>Ethambutol </li></ul></ul><ul><ul><li>Pyrazinamide </li></ul></ul><ul><li>Second-line Drugs </li></ul><ul><ul><li>Cycloserine </li></ul></ul><ul><ul><li>Ethionamide </li></ul></ul><ul><ul><li>Levofloxacin </li></ul></ul><ul><ul><li>Moxifloxacin </li></ul></ul><ul><ul><li>Gatifloxacin </li></ul></ul><ul><ul><li>P -Aminosalicylic acid </li></ul></ul><ul><ul><li>Streptomycin </li></ul></ul><ul><ul><li>Amikacin/kanamycin </li></ul></ul><ul><ul><li>Capreomycin </li></ul></ul><ul><ul><li>Linezolid </li></ul></ul>
  77. 77. <ul><li>-BCG </li></ul>PREVENTION CHALLENGES WHO Jan 07 “ BCG vaccine should not be used in children known to be HIV-infected”
  78. 78. THANK YOU