This document provides information on the diagnosis and treatment of tuberculosis. It discusses methods for collecting and analyzing sputum samples under microscopy and culture to identify Mycobacterium tuberculosis. It also summarizes techniques for assessing drug resistance and extra-pulmonary tuberculosis diagnosis. Treatment involves short course multidrug regimens and directly observed therapy programs.

1. TUBERCULOSIS
Dr.Riyaz Sheriff

2. PULMONARY TUBERCULOSIS
 Bacillary shedding  Sputum
 Caseation : Abundant
 Organized lesions: Scanty
 Sample best collected early morning ..
 Best to collect 2 samples
 Spot sample
 Early morning sample
 Laryngeal swab
 Bronchial washings
 Gastric lavage (in children )

3. Microscopy
 Direct or concentrated smears of sputum
 Reliable method for diagnosis
 Do not reuse the slides
 Smears prepared from thick and purulent part
of sputum
 Smears dried, Heat fixed and stained by Ziehl-
Neelsen technique

4. Diagnosis by microscopy
 Minimum 10,000 AFB/ ml of sputum should be present
 300 fields to be examined before giving a report
 Positive report can be given if 2 or more bacilli are seen
 Grading
 Microscopy : Presumptive evidence
 Saprophytic Mycobacteria can be misleading
 Saprophytic species  Stain uniformly (No beading/ No Barred
appearance), only acid fast. Not alcohol fast

5. Saprophytic mycobacteria
 Seen in
 Tap water
 Rubber tubes
 Cork
 Barks
 Not seen in respiratory secretions
 Seen in gastric aspirates
 Feces
 Urogenital specimens

6. Grading of ziehl –neelsen smears
NUMBER OF AFB NO.OF FIELDS REPORT
0 1000 Negative
1-9 100 SCANTY
10-99/ 100 FIELDS 100 1+
1-10/ FIELD 50 2+
10 or more/ FIELD 20 3+

7. Fluorescent microscopy
 Useful in screening larger number of smears
 Smears stained with Auramine Phenol or
Auramine rhodamine fluorescent dyes
 Examined under ultraviolet illumination
 Seen using 40X objective
 Bacilli appear as bright rods in a dark
background

8. Fluorescent microscope

9. Fluorescent microscope

10. Afb in fluorescent staining

11. Concentration methods
 For microscopy
 For culture
 For animal inoculation

12. For microscopy only
 Procedure kills the bacilli without altering the
shape or staining reaction
 Methods
 Antiformin
 Sodium carbonate
 Hypochlorite
 Detergents
 Tergitol
 Flotation
 Hydrocarbons
 Autoclaving

13. Homogenisation & Concentration
 Petroff’s method
 Simple
 Sputum incubated with equal volume of 4%NaOH @ 37°C with frequent
shaking
 Wait for 20minutes or till the mixture becomes clear
 Centrifuge at 3000rpm for 20mins
 Discard the supernatent
 Neutralize the sediment with N/10Hcl
 This sediment is used for smear, culture and animal inoculation

14. Method 2
 Sterile solution - 20gms cetrimonium bromide and 40 g of
NaOH added to litre of distilled water
 Sputum treated with equal amounts of sterile solution
prepared
 Contents mixed with cotton swab and allowed to stand for
5mins
 0.2ml of inoculum smeared firmly on IUAT-LJ medium with
KH2PO4 per litre
 Same swab use for inoculating another medium

15. Homogenization
 NaOH
 Dilute acids
 6%Sulphuric acid
 3%Hcl
 5%Oxalic acid
 N- acetyl cysteine with NaOH
 Pancreatin
 Desogen
 Zephiran
 Cetrimide

16. Culture
 Sensitive
 Needs only 10-100bacilli / ml of sputum
 2 bottles of LJ medium are inoculated for every sample`
 Drug sensitivity can also be done if needed
 First examination after 4 days  Rapid growers, Contamination &
Fungus
 Any slow growing, Non pigmented , Niacin positive, Acid Fast Bacilli :
M.tuberculosis
 Recent trends : automated cultures (BACTEC 460), Identification using
nucleic acid probes  report can be issued in 2-3 weeks

17. Sensitivity testing
 In an era of Multidrug resistance Sensitivity
testing helps in instituting treatment.
 Types
 ABSOLUTE CONCENTRATION METHOD
 RESISTANCE RATIO METHOD
 PROPORTION METHOD
Media is inoculated with serial concentration of drugs and
minimum inhibitory concenration (MIC) is calculated
•Two sets of media containing graded concentrations of
the drug are inoculated.
•One set is inoculated with test strain and another set is
inoculated with a standard strain of known sensitivity
•Average sensitivity of strain .
•Considers the fact that a population of bacilli will
contain varying resistance

18. Animal inoculation
 Guinea pigs : 2
 12 weeks old
 Weighed before inoculation
 Intramuscular inoculation
 Animal weighed at regular intervals
 Progressive loss of weight  INFECTION
 Tuberculin : POSITIVE
 One animal killed at 4 weeks.
 Caseous lesion at the site of inoculation
 Pus filled localized lesions
 Draining lymph node enlargement

20.  Spleenomegaly with necrotic areas
 Tubercles in peritoneum, lung
 No lesions in the kidney
 Autopsied lesions stained using Ziehl Neelsen
staining : to R/O Y.pseudotuberculosis
 If no positive findings the second animal is
killed at 8 weeks
Animal inoculation

21. Genes to our rescue!
 Polymerase chain reaction
 Ribosomal RNA amplification
 Ligase chain reaction
 RFLP

22. Immunodiagnosis
 Serology : NOT USEFUL in tuberculosis
 Tuberculin testing : age old method: Does not
diagnose infection
 Detection of antibody to Mycobacterial lipo-
arabinoannan may be useful

23. Tuberculin testing : Mantoux test
 0.1ml of Purified Protein Derivative (PPD) containing 5 Tuberculin
Units (TU)
 Injected INTRADERMALLY in flexor aspect of forearm using a tuberculin
syringe
 Injection site examined after 48-72hours
 Induration measured transversely to long axis of forearm.
 >10MM  POSITIVE
 <5MM  NEGATIVE
 6-9MM  EQUIVOCAL
 PPD dose of 1TU is used when extreme hypersensitivity is suspected
 PPD dose of 10 or 100 TU is used when test with 5TU is negative

25. Other tests
 Heaf Test:
 Useful in screening and surveys
 Multiple puncture technique
 Not diagnostic
 Tine test :
 Disposable prongs containing dried PPD for
individual testing

26. Heaf test

27. Tine test

28. Interpretation of tuberculin tests
 POSITIVE
 Infection with tubercle bacillus / BCG vaccination
 May or may not have clinical disease
 4-6 weeks post exposure
 If no further exposure tuberculin allergy wanes by 4-5 years
 FALSE POSITIVE
 Infection with atypical mycobacteria
 TWO STEP TESTING
 Repeated tuberculin testing  enhances intensity of response in a reactive
individual
 Useful in patients with equivocal results
 Second testing should be done on a different site

29. Interpretation of tuberculin tests
 NEGATIVE
 No contact with tubercle bacilli
 FALSE NEGATIVE
 ANERGY
 Inactive PPD
 Faulty injection Technique
 Miliary tuberculosis
 Post viral infections : Measles
 Lymphoreticular malignancies
 Sarcoidosis
 Severe malnutrition
 Immunosuppressive therapy
 Impaired CMI

31. EXTRAPULMONARY TUBERCULOSIS
Bacilli are fewer compared to pulmonary disease
Tip: CSF from tuberculous meningitis forms
spider web clot on standing
Bone marrow , Liver biopsy From miliary TB :
Culture
Blood from HIV coinfection :Culture
Pus from tuberculous abscess : Positive on smear
and culture
Pleural effusion and other exudates collected in
citrated tubes to prevent coagulation

32. EXTRAPULMONARY
TUBERCULOSISIn the absence of bacterial contamination
exudates can be used for culture directly
If bacterial contamination is present
concentration techniques are employed before
inoculating in culture medium .
In renal Tuberculosis the excretion of bacilli is
intermittent , Hence 3-6 morning samples of
urine are collected
Centrifuged at 3000rpm for 30mins
Sediment use for culture

33. PROPHYLAXIS : BCG
 BCG = Bacille Calmette Guerin
 Attenued strain of M.bovis by 239 serial subcultures in
glycerine – bile –potato medium over a period of 13 years!!
 Immunity lasts for 10-15 years
 Given immediately after birth
 Efficacy varies
 Protection against meningitis , disseminated tuberculosis

34. PROPHYLAXIS : BCG
 Should not be given to babies with active HIV
 Should not be given to babies born to AFB positive
mothers, Given after a course of preventive chemotherapy
 Stimulates immune system
 Some protection against leprosy and leukemia
 Adjuvant in therapy of some malignancies

35. Chemotherapy: anti-tubercular drugs
 Preventive chemotherapy
 Isoniazid :
 Latent TB
 High risk population
 Infants of mothers with active infection
 Children living in active TB house hold
5mg/kg body weight daily for 6-12 months

36. Chemotherapy: anti-tubercular drugs
 Bacteriocidal
 Rifampicin (R)
 Pyrazinamide (Z)
 Isoniazid (H) : Effective against replicating bacilli
 Streptomycin (S) : effective against extracellular
bacilli
 Bacteriostatic
 Ethambutol (E)
 Short course regimens (HRZE) : 6-7 months
 Intensive phase : 2months
 Continuation phase (HR) : 4 months

37. Drug resistance
 Mutations
 Multidrug therapy
 Not followed properly  Bacteria has become resistant
 Primary resistance : infected with resistant bacilli
 Secondary resistance : Initially sensitive but becomes resistant during course
of treatment
 Secondary resistance more common
 MDR-TB : resistance to Rifampicin & Isoniazid
 Second line drugs : Quinolones , Aminoglycosides , Macrolides, PAS,
Cycloserine etc
 XDR – TB : MDR + resistance to fluroquinolones and injectables

38. Rntcp & dots
 Revised National Tuberculosis Control
Program – 1933
 Directly Observed Treatment, Short course
Chemotherapy
 DOTS plus in XDR cases