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Lab diagnosis of tb dr mostafa lecture

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concise discussion about the available lab diagnosis of TB in developing and developed countries.

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Lab diagnosis of tb dr mostafa lecture

  1. 1. Concise Laboratory Diagnosis of Tuberculosis Infection (TB) Dr Mostafa Mahmoud, MD, Ph D Consultant Microbiologist Assist. Prof. of Medical Microbiology & Immunology
  2. 2. Pulmonary, 70% Extrapulmonary, 21% Both, 9% Pleural, 18% Lymphatic, 42% Bone/joint, 11% Genitourinary, 5% Meningeal, 6% Other, 12% TB Cases by Form of Disease, United States, CDC, 2005 Peritoneal, 6% Clinical Presentation of TB: Extrapulmonary  Incidence/site may vary  TB can involve any organ  More common in HIV/TB (co-infection)
  3. 3. Introduction  80% of all TB cases worldwide are from 22 high- burden countries (India, China, Indonesia , Nigeria, Bangladesh, South Africa, Pakistan, Ethiopia, Philippines, Congo, Vietnam, Tanzania, Brazil, Uganda, Thailand, Mozambique, Myanmar, Afghanistan, Cambodia) arranged in a descending order by the number of cases.
  4. 4. Lab Diagnosis of TB. 1- Direct Microscopy Smearing (Staining) (AFB stains and Fluorescent) 2- Culture: (Solid and liquid media also for measurement of drug susceptibility testing DST for resistance). 3- Gamma interferon Release Assays ( Spot test and gold????) 4- Molecular testing (PCR and other NAA) 5- Serology 6- Histopathology.
  5. 5. Samples  Type of sample: Sputum (resp. sample), BAL CSF (spinal/para-spinal/intra-cerebral), gastric washings (Aspirate), lymph nodes (tissues), urine, faeces, blood
  6. 6. SPECIMEN QUALITY  Now 2 specimens are used; before three specimens on three or 2 successive days  Early morning specimens (First thing out!)  Adequate specimen volume (>7 ml)  Sputum not saliva (epithelial cells > 25/ LPF = Saliva)  Condition of specimen (pH, etc.)  Appropriate specimen container  Complete and accurate information  Proper packaging (i.e. US Postal Service)  “Prompt” delivery to the Lab.  Proper documentation.
  7. 7. Transfer to laboratory  Within 24 h (or 1 working day, max 48h) Minimise overgrowth Maintain AFB character  Potentially infected clinical sample Routine procedure
  8. 8. 1- Direct AFB Slide Examination  Least Sensitive AFB Test (20% to 80%) increase with advance of the TB infection.  Requires 100,000 AFB/ml  Requires an adequate specimen > 5 ml  Negative smear – Does not rule out AFB  Positive smear – AFB present (viability?)  AFB Positive = Mycobacterium spp., etc.  Help support TB diagnosis  Quantitation: Positive/Positive Few/ Negative
  9. 9. Formats: 1. Conventional Microscopy with sensitivity of 32 to 94%. 2 techniques:- i- Hot AFB staining (Ziehl-Neelsen) Method the most common ii- Cold AFB staining (Kynon staining) 2- Fluorescent Microscopy with sensitivy of 52% to 97% by Auramine staining (more sensitive and less cumbersome (low power) than other 2 previous ones. However, less specific and needs confirmation by Z-N staining). - LED microscopy in now recommended to replace the fluorescent microscope in all situations. - Concentrated smears are more sensitive than direct one.
  10. 10. Ziehl- Neelsen Stain
  11. 11. Steps of Ziehl- Neelsen Staining
  12. 12. Auramine stained smear
  13. 13. Smear positive AFB by Ziehl-Neelsen
  14. 14. Recording Sputum Smear Microscopy Results Number of Acid- fast Bacilli (AFB) # of Oil Immersion Fields Examine Reported as: No AFB Per 100 fields No AFB seen (No AFB per 100 fields) 1-9 AFB Per 100 fields Scanty, record exact figure (1-9 AFB per 100 fields) 10-99 AFB Per 100 fields 1+ (10-99 AFB per 100 fields) 1-10 AFB Per field 2+ (1-10 AFB per field in 50 fields) More than 10 AFB Per field 3+ (>10 AFB per field in 20 fields)
  15. 15. 2- Culture methods: solid versus liquid Solid: on Lowenstein- Jensin (LJ) slopes. 7H11 Middlebrooks slopes or plates. On LJ media it takes 12 weeks incubation. Liquid: Early attempts high contamination. Recommended by WHO on liquid Middlebrooks (7H9) broth media Automated liquid systems -Bactec MIGT 960; in common use nowadays – Bactec 460TB; old and use radioactive C 14 - ESP blood culture system More Sensitive and Rapid. Contamination with non-tuberculous MB can give false results.
  16. 16. Solid LJ media
  17. 17. Middlebrooks media liquid and solid
  18. 18. Automated Bactec MGIT 960
  19. 19. Advantages of AFB Culture and Isolation  More sensitive than slide Microscopy (80%-90%)  Requires only 10 AFB/ ml of specimen  AFB Growth in 1-2 weeks (Bactec)  Genetic Probes and HPLC Rapid ID  Fewer AFB/ml – Delayed growth  “AFB Growth” required for other tests e.g. species identification and drug susceptibility.  Mixed cultures require subcultures
  20. 20. 3- Gamma interferon Release Assays (Immunodiagnostic) tests  Used for detection (after in vitro stimulation) of either:  Interferon γ (QuantiFERON-TB Gold)  Activated specific T-cells (T-SPOT.TB) Standard under development Which patients? Mostly for latent TB How long should it take? Around 1 day Who provides it? What do the results mean and who interprets them?
  21. 21. Explanation of immunodiagnostic assays:
  22. 22. Interferon gamma release assays
  23. 23. Quantiferon and T-Spot TB kits
  24. 24. Advantages of gamma interferon release assay:  One visit test  Not affected by vaccination with BCG.  Can diagnose latent and active disease.
  25. 25.  1- From mycobacterial cultures for species identification: i- Nucleic Acid hybridization (Gen-probe) for 16S rRNA from culture isolates ii- Line Probe assays (e.g. Hain Strips) it is reverse hybridization. iii- Fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) (TB PNA FISH) sensitivity 84-97%. iv- PCR with or without sequencing v- Xpert MTB/RIF (Cepheid) new fully automated but costly technique for identification and DST. 4- Molecular diagnosis of Mycobacterial
  26. 26. vi- Other NAAT for typing: e.g. LCR, Spoliotyping, RFLP etc. 2- Directly from clinical specimens:  MTD (Gen-probe) or Amplicor (Roche ) for MTB PCR
  27. 27. Advantages of molecular test: More rapid results within hours.  high specificity 98% - 100 %. However, Less specific giving false positive and negative results in clinical samples. Disadvantages:  High costs.
  28. 28. GenXpert System Cepheid
  29. 29. 5- Serology  For detection of either antigens or antibodies.  Little value in laboratory diagnosis.  Variability and cross-reactivity in results i.e. low specificity and sensitivity.  Not distinguishing active from latent infection  Formats: - ELISA (82 sensitivity and 86% specificity). - Detection of lipoarabinomannan (LAM) antigen
  30. 30. Recent Technologies:  1- MALDI TOF (Matrix Assisted Laser Desorption Ionization Time of Flight MS)  Very rapid technique (< 1 hour)  2- GenXpert (Cepheid) System  Rapid  Simple  All reagent in one cartridge  Can be used in general lab no need for spate rooms  Used for clinical specimens  Gives specie identification and drug susceptibility.
  31. 31. What test formats available in MOH  1- Ziehl-Neelsen Stain  2- Kynon Cold Stain  3- GenXpert (Cepheid)  4- Roche Amplicor PCR and sequencing
  32. 32. References:  Imperial College Healthcare (NHS)  Recent advances in the laboratory diagnosis of tuberculosis, Rommy Teran1, Jacobus H. de Waard2  Parson LM et al. 2011. Laboratory Diagnosis of Tuberculosis in Resource-Poor Countries: Challenges and Opportunities. CLINICAL MICROBIOLOGY REVIEWS, Apr. 2011, p. 314–350.  Anochie PI et al.  Recent advances in the diagnosis of Mycobacterium tuberculosis; Review. www.germs.ro • GERMS 2(3) • September 2012 • page 110

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