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Similar a LaboratorydiagnosisofTB.pptx(20)



  2. CLASSIFICATION OF MYCOBACTERIA Mycobacterium tuberculosis complex refers to a genetically related groups of Mycobacterium species that can cause TUBERCULOSIS [TB] in humans. It includes; Mycobacterium tuberculosis, Mycobacterium bovis [M.bovis, subsp bovis, M.bovis subsp.caprae and M.bovis BCG]
  3. SPECIMEN COLLECTION In every clinical Microbiology sample collection “Results are as good as Specimen” Good quality sample is very important. Sputum samples of good quality collected in wide mouth sterile containers. Quantity sufficient Extra pulmonary samples collected in sterile containers /syringes.
  4. Mucoid Sample Purulent Sample Bloody Sample Salivary Sample
  5. DIAGNOSIS OF TUBERCULOSIS Smear Microscopy AFB Culture Manual & Liquid Sensitivity Molecular Diagnostic
  6. In low income and high tuberculosis prevalence countries, sputum smear microscopy is the only cost- effective tool for diagnosing patients with infectious tuberculosis and to monitor their progress in treatment. Sputum smear microscopy is a simple, inexpensive, appropriate technology method which is relatively easy to perform and to read. Classical Ziehl-Neelsen stain used-AFB SMEAR MICROSCOPY
  7. Smear prepared from thick purulent parts of samples. Size of smear should be 3cmx2cm At least 300 fields examined Smear is positive in samples which contain 5000- 10000 bacteria /ml Sensitivity ranges from 25%-65%  Sensitivity increases by examination of more than one smear SMEAR MICROSCOPY
  8. SIZE OF SMEAR 2 x 3 1 x 2 Uniform Smear
  9. Good Evenness Smear Uneven Smear Good Thickness Smear Too Thick Smear Too Thin Smear
  10. Smears reported as Positive or Negative Quantity of AFB observed should be noted Factors influencing smear sensitivity are type of specimen, staining technique, experience of reader Laboratory Quality Control important ZN STAINING
  11. FLUORESCENCE MICROSCOPY Fluorochrome stain used Can be examined at lower magnification(40 X) Rapid but more false positive LED fluorescence microscopy has been evaluated- rapid and good results, lower cost LED attachment to microscope Primo Star iLED from Carl Zeiss
  13. DISADVANTAGES OF SMEAR MICROSCOPY Needs a large no of bacilli per ml of specimen to be detected positive Cannot differentiate between dead and live bacilli Cannot differentiate between Mtb and NTM No idea of drug resistance
  14. AFB CULTURE GOLD STANDARD Provides definitive diagnosis of TB Pure growth of mycobacteria to do speciation and drug sensitivity. Technically demanding and complex High level of Biosafety needed
  15. AFB CULTURE BY L.J. MEDIA [SOLID] Detection of 10-100 viable bacilli/ml of specimen Specimens have to be decontaminated before inoculation to remove the normal bacterial flora. Solid culture – Conventional LJ method. Mycobacteria slow growing and hence take 2-8 weeks to grow LOWENSTEIN JENSEN [L.J. ] MEDIA
  16. LIQUID CULTURE Many Commercial systems available- BACTEC systems MGIT960, BacT/ALERT 3D system Liquid culture yield significantly rapid results than solid media and isolation rates for mycobacteria are higher Liquid media- Middle brook 7H9 media used MGIT system( Mycobacterial growth indicator tubes) contains a modified Middle brook 7H9 broth with a fluorescence quenching based oxygen sensor. Growth of mycobacteria leads to oxygen depletion and indicator fluoresces brightly Cultures positive in 10-14 days
  18. DRUG SENSITIVITY FOR AFB Sensitivity to first and second line drugs available Expensive High degree of technical expertise and lab infrastructure required Rigorous quality control needed
  19. NON COMMERCIAL METHOD MODS [Microscopic observed drug susceptibility]  A micro colony method in liquid culture , based on inoculation of specimens into drug free and drug containing media, followed by microscopic examination of early growth . Recommended as direct or indirect tests for rapid screening of patients suspected of having MDR TB
  20. CRI ( Colorimetric redox indicator) Indirect testing methods based on the reduction of a coloured indicator added to liquid culture medium on a microtitre plate after exposure of M. tb strains to anti TB drugs in vitro
  21. NRA (Nitrate reductase assay)  A direct or indirect method on solid culture based on the ability of M. tuberculosis to reduce nitrate, which is detected by a colour reaction
  23. MOLECULAR TEST Genotypic methods have considerable advantage of speed, standardization of testing and reduced requirement for Biosafety 1. LINE PROBE ASSAY ( HAINS TEST) 2. GENEXPERT
  24. 1. LINE PROBE ASSAY ( HAINS TEST) Simultaneous identification for M.tuberculosis complex Molecular assay for the detection of resistance to INH & RIF of M.tuberculosis complex By detection of most significant mutations to – inhA, RpoB and the katG genes Based on DNA strip technology Can be done from positive cultures (from MGIT, BacT/ALERT bottles or LJ) Pulmonary samples which are smear +ve can be done directly
  25. Detection of multiple genes responsible for the antibiotic resistance & Simultaneous recognition of missing wild type gene Also Available for Second secondline and identification of some strain of NTM Limitations of Genotype MTBDRplus Needs preprocessing of samples. Needs a PCR set up Technically demanding Panic of contamination Special infrastructure required Needs dedicated staff and space.
  26. 2. GENEXPERT [CBNAAT] The Xpert MTB/RIF is a cartridge based nucleic acid amplification test , automated diagnostic test that can identify Mycobacterium tuberculosis (MTB) DNA and resistance to Rifampicin (RIF) by Nucleic Acid Amplification Test(NAAT). SAMPLES; Pulmonary samples( Sputum, BAL ) Extra pulmonary samples [Lymph node tissue and aspirates, CSF, Pus , Gastric lavage and aspirates ( in children) & Other Tissues]
  27. Pulmonary samples - Xpert MTB/ Rif Sensitivity Status Sensitivity % Smear +ve culture +ve 98 Smear –ve culture +ve 68 People with HIV 79 People without HIV 86 Extra pulmonary samples Xpert MTB/Rif - sensitivity and specificity Samples Sensitivity % Specificity % Lymphnode tissue and aspirate 84.9 92.5 CSF 79.5 98.6 Pleural fluid 43.7 98.1 Gastric lavage and aspirations 83.8 98.1 Other tissue 81.2 98.1