TB-Sputum AFB Test

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TB-Sputum AFB Test

  1. 1.  Demonstration of AFB by sputum microscopy is the most confirmatory test for pulmonary tuberculosis, but one has to be sure that sputum has come from lungs and it is not just saliva.  Single direct smear miss about 25% of sputum positive cases  Simple, inexpensive, appropriate technology which is relatively easy to perform and to read, yields timely results with very high sensitivity of detection of tubercle bacilli.
  2. 2.  Early diagnosis of mycobacterial infections.  Confirm acid fast nature of organism.  Monitor effectiveness of treatment.  May be determinant of performing other tests such as culture or susceptibility testing.
  3. 3.  Severe cough for 2 or more weeks (often involving       blood loss) Weight loss Night sweats Weakness or fatigue(tiredness) Chills Fever Pain in the area infected.
  4. 4.  Under NTP conditions, the IUATLD recommends collecting 2 sputum samples “on the SPOT-early MORNING” preferably within 2 days, from each person presenting at health centre, with respiratory symptoms of more than 3 weeks duration.  Volume of sputum: 3ml-6ml(minimum acceptable amount:2ml)  SPOT specimen when TB suspects present at the health centre.  MORNING specimen sputum produced within 1 or 2 hours after rising.
  5. 5.  If sputum are collected within the same 24 hour period, a minimum of 8 hours between specimen is required.  Sputum smear positive tuberculosis a person presenting with respiratory symptoms with at least one positive sputum smear microscopy examination.  Detects about 80% of TB suspects ultimately positive on sputum smear examination with the 1st specimen, and additional 15% with the 2nd specimen .
  6. 6.  5-10 ml  Thick and mucous, but may be fluid with pieces of purulent material.  Colour varies from opaque white to green, but reddish to brown when blood is present.
  7. 7.  Primary stain binds cell wall mycolic acid.  Intense decolorization does not release the primary stain from the cell wall.  Colour of AFB based on primary stain.  Counter stain provides contrasting background.
  8. 8.  Transmitted light: Carbol fuchsin staining.[contrast red colour AFB on blue background]  Fluorescence : Auramine staining[contrast light/dark]
  9. 9. PROCEDURE  Make the smear on a clean glass slide, air dry and keep it fixed.  Add conc. Carbol fuchsin, kept for 5-7 minutes, with intermittent heatings (till fumes arise), wash with water.  Add 20% H2SO4, kept for 5 minutes, wash with water.  Add methylene blue, kept for 3 minutes, wash with water.  Air dry and observe under oil immersion objective of microscope.
  10. 10. ZN STAINING
  11. 11. ZN STAINING
  12. 12. AURAMINE STAINING
  13. 13.  A well stained ZN smear shows strong red AFB against a weak blue background.  Examine 100 fields with the bright field microscope, 1000X magnification.  Declare the smear negative if no AFB are found.  For high positives examination of 20-30 fields may suffice.
  14. 14. No. Examination(No. of bacilli) No. of fields Grading 1 0 300 -ve 2 2-3 300 Doubtful, repeat smear. 3 1-9 100 1+ 4 1-9 10 2+ 5 1-9 1 3+ 6 >10 1 4+
  15. 15.  Acid fast particles and other micro- organisms(waxes, precipitate of stain, nocardia, environmental mycobacteria, spores of bacillus subtilis, fibres and pollens, food particles, yeast and artifacts like scratch on slide)
  16. 16.  Inadequate sputum collection, failure to select suitable sputum particles for making smear, poor smear preparation and inadequate examination of smears.
  17. 17.  Specific and confirmatory test for pulmonary     tuberculosis. It is the test for infectiousness of the patient. Sputum examination is important to monitor progress during chemotherapy. It helps to detect drug failure and development of resistance in patients in anti tubercular treatment(fall and rise phenomenon) It is most cost effective investigation.
  18. 18.  It is less sensitive investigation compared to radiology. Since number of bacilli required for demonstration of AFB in sputum is quite enormous(10,000 bacilli/ml3), the cases excreting less number of bacilli may not be diagnosed by microscopy alone. Similarly cases of pleural effusion and hilar lymphadenitis may also be missed.  Does not differentiate between living and dead bacilli.  Typing of bacilli is not possible.  False positive result can mislead in diagnosis.
  19. 19.  Sensitivity can be increased by concentration method, processing sample with NALC-NAOH method require expertise, but can detect 15-20% more cases.

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