1. Laboratory Diagnosis of Tuberculosis By Dr. Faten Aly Shoukry, Ph D Consultant & Head of Microbiology Department, Abbassia Chest Hospital

2. Types of Tuberculous Infections I- Pulmonary Infection II- Extrapulmonary infections: a-TB Pleural effusion a-TB Pleural effusion b- Tuberculous meningitis b- Tuberculous meningitis c- Millary tuberculosis. c- Millary tuberculosis. d- Renal & urogenital tuberculosis. d- Renal & urogenital tuberculosis. e- Bone &joint tuberculosis. e- Bone &joint tuberculosis. f- TB entritis. f- TB entritis.

3. Types of specimens: -Sputum. - BAL. -Pleural effusions - Urine - Stool -CSF -Aspiration ( gastric – cold abscess) - Blood in case of haematogenous TB

4. Laboratory Diagnosis 1- Sputum smears stained by Z-N stain Three morning successive mucopurulent sputum samples are needed to diagnoise pulmonary TB. Advantage: - cheap – rapid Advantage: - cheap – rapid - Easy to perform - Easy to perform - High predictive value > 90% - High predictive value > 90% - Specificity of 98% - Specificity of 98%Disadvantages: - sputum ( need to contain 5000-10000 AFB/ ml.) - sputum ( need to contain 5000-10000 AFB/ ml.) - Young children, elderly & HIV infected persons may not produce cavities & sputum containing AFB. - Young children, elderly & HIV infected persons may not produce cavities & sputum containing AFB.

5. Interpretation of sputum stained by Z N Stain (WHO ) More than 10 bacilli / field ------- +++ From 1 – 10 bacilli / field ------- ++ From 10 – 99 bacilli / 100 fields ----- + From 1 -9 bacilli/100 fields ------ write the no. No bacilli seen ---------- negative

7. 2- Detecting AFB by fluorochrome stain using fluorescence microscopy: The smear may be stained by auramine-O dye. In this method the TB bacilli are stained yellow against dark background & easily visualized using florescent microscope. Advantages: Advantages: - More sensitive - More sensitive - Rapid - RapidDisadvantages: - Hazards of dye toxicity - Hazards of dye toxicity - more expensive - more expensive - must be confirmed by Z-N stain - must be confirmed by Z-N stain

8. 3- Cultures on L J media Lowenstein –Jensen medium is an egg based media with addition of salts, 5 % glycerol, Malachite green & penicillin. Advantages: - Specificity about 99 % - More sensitive ( need lower no. of bacilli 10-100 / ml) - More sensitive ( need lower no. of bacilli 10-100 / ml) - Can differentiate between TB complex & NTM using biochemical reactions - Can differentiate between TB complex & NTM using biochemical reactions - Sensitivity tests for antituberculous drugs - Sensitivity tests for antituberculous drugs ( St, INH, Rif., E) ( St, INH, Rif., E) Disadvantages: Slowly growing ( up to 8 weeks )

10. Recent Methods for Diagnosis I – BACTEC 460 ( rapid radiometric culture system ) specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C 14 – labelled palmitic acid & PANTA antibiotic mixture. specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C 14 – labelled palmitic acid & PANTA antibiotic mixture. Growing mycobacteria utilize the acid, releasing radioactive CO 2 which is measured as growth index (GI) in the BACTEC instrument. Growing mycobacteria utilize the acid, releasing radioactive CO 2 which is measured as growth index (GI) in the BACTEC instrument. The daily increase in GI output is directly proportional to the rate & amount of growth in the medium. The daily increase in GI output is directly proportional to the rate & amount of growth in the medium.

11. The PANTA antibiotic mixture P ---- Polymyxin B P ---- Polymyxin B A ---- Amphotericin B A ---- Amphotericin B N ---- Nalidixic acid N ---- Nalidixic acid T ---- Trimethoprim T ---- Trimethoprim A ---- Azlocillin A ---- Azlocillin The antibiotic mixture inhibits the growth of contaminating bacteria.

12. Advantages : - Rapid (mycobacteria can be detected within 12 days.) - Determining drug susceptibility. - Differentiating between TB complex & NTM by NAP test. - - Specificity is very high Disadvantages: - Expensive - Expensive - Hazards of using radioactive material. - Hazards of using radioactive material.

13. NAP Test Members of M.tuberculosis complex do not grow in the presence of p-nitro-∞-acetylamine Members of M.tuberculosis complex do not grow in the presence of p-nitro-∞-acetylamine-ß-hydroxypropiophenone. If 5 ug of NAP is added to actively growing culture in 12B medium vial the growth of M. tuberculosis complex is inhibited while Mycobacteria other than tuberculosis (NTM) do not demonstrate significant inhibition. If 5 ug of NAP is added to actively growing culture in 12B medium vial the growth of M. tuberculosis complex is inhibited while Mycobacteria other than tuberculosis (NTM) do not demonstrate significant inhibition.

15. II Mycobacteria Growth Indicator Tube (MGIT) Tube contains modified Middlebrook 7H9 broth base with OADC enrichment & PANTA antibiotic mixture. All types of clinical specimens, pulmonary as well as extra-pulmonary ( except blood ) could be cultured on this type of media.

16. The OADC supplement O ----- Oleic acid ( Metabolic stimulant) O ----- Oleic acid ( Metabolic stimulant) A ----- Albumin ( to bind toxic free fatty acid ) A ----- Albumin ( to bind toxic free fatty acid ) D ---- Dextrose (Energy source ) D ---- Dextrose (Energy source ) C ----- Catalase ( Destroy toxic peroxides that may be present in the medium ) C ----- Catalase ( Destroy toxic peroxides that may be present in the medium )

17. Principle of the procedure: Principle of the procedure: A fluorescent compound (which is sensitive to O 2 ) is embeded in silicone on the bottom of the tube. The actively respiring microorganisms consume the oxygen & allow the fluorescence to be observed using UV trans-illuminator lamp.

18. The MGIT 960 System The MGIT 960 system is a non-radiometeric automated system that uses the MGIT media & sensors to detect the fluorescence. The MGIT 960 system is a non-radiometeric automated system that uses the MGIT media & sensors to detect the fluorescence.Advantages: -The system holds 960 plastic tubes which are continuously monitored. - Early detection as the machine monitoring & reading the tubes every hour.

19. III Polymerase Chain Reaction (PCR) & Gene probe Nuclic acid probes & nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells. Nuclic acid probes & nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells.Advantages: - Rapid procedure - High sensitivity (1-10 - Rapid procedure - High sensitivity (1-10 ( 3 – 4 hours) bacilli / ml sputum) ( 3 – 4 hours) bacilli / ml sputum)

21. Disadvantages: - Very expensive. - Require specialist training & equipments. - False positive results. - Can not differentiate between living & dead bacilli.

22. IV FASTplaque TB Test - Patient’ s sputum is mixed with myco-bactriophage. - A virucide is added which destroy any phages outside the TB bacilli. - Lysis of cells & release of phages after replication within the tubercle bacilli. - Non-pathogenic mycobacteria are added & the sample incorporated in agar mixture( over night incubation) - Zones of clearing indicate that patient’ s sputum contained viable M. tuberculosis.

24. Interferon –γ Tests An in vitro T-cell-based assays tests for diagnosis of latent TB infection : - QuantiFERON TB gold Test - QuantiFERON TB gold Test - T-Spot Test. - T-Spot Test. These whole-blood assays measure IFN-γ production by previously sensitised lymphocytes in response to M.tuberculosis- specific protein antigens ESAT6 and CFP-10 These whole-blood assays measure IFN-γ production by previously sensitised lymphocytes in response to M.tuberculosis- specific protein antigens ESAT6 and CFP-10

25. Some studies have shown that: -Compared with TST these tests have higher specificity. - -Correlate better with exposure to tuberculosis. - -Have less cross-reactivity with the BCG vaccine & environmental mycobacteria. - NB: Yet there is no evidence for the use of these tests in young children at present. NB: Yet there is no evidence for the use of these tests in young children at present.

26. Evaluation of different methods of diagnosis As regards the time: The MGIT had the shortest mean time to positivity at 13.3 days, compared with 14.8 days for the BACTEC 460 system & 25.6 days for L J medium. The MGIT had the shortest mean time to positivity at 13.3 days, compared with 14.8 days for the BACTEC 460 system & 25.6 days for L J medium.

27. As regards the no. of culture yield: The best yield, was with BACTEC 460, followed by BACTEC MGIT 960, & then with L J medium. The best yield, was with BACTEC 460, followed by BACTEC MGIT 960, & then with L J medium. As regards contamination rate: As regards contamination rate: L J medium (17%) had the highest contamination rate (Tortoli E, Cichero P,Et al. 1999) then the MGIT 960 ( 10.0% ) Compared with radiometeric system (3.7%)

28. Thank you