laboratory daignosis of pulmonary tuberculosis and what happens in the lab and how to deal with the specimens and how to know the positive frome negative results

1. PTB
Diagnosis

2. Laboratory diagnosis of p.TB is the
most challenging
For three reasons :-
1 ) collection of sputum sample from the oral cavity which get contaminated
with oral bacteria so the sample has to be decontaminated first.
2 ) slow growing … so culture must be incubated for up to 6 weeks to recover
the microorganism.
3 ) highly infectious … so all the cultivation has to be done in a biosafety lab.

5. The sample is sputum 5ml

7. Decontamination of sputum
1 ) An equal volume of( Noah )is added to sputum
2 ) Mixed by mixture to emulsify
3 ) Then left to decontaminate for 25 min.

8. Concentration of mixture
1 ) The sputum & Naoh mixture is centrifuged at 3000 g for 15 min.
( 3750 revolution per min. )
2 ) The supernatant is decanted & the concentrated deposited is
resuspended by phosphate puffer to neutralize sodium hydroxide .
3 ) And its mixed & centrifuged again .

9. The final buffer deposit is go either to …. Or ….
A ) Staining by acid fast Or B ) Culture on liquid or solid media

10. A ) The staining method
The smear is made of sputum deposit is stained
by one of two methods
1 ) auramine phenol fluorescence stain
2 ) ziehl-Neelsen stain ( ZN stain ).
The cell wall of mycobacteria contain a highly
concentration of lipid making them waxy &
hydrophobic & impermeable routine stain such
as the gram’s stain
They are also resistant to acid & alcohol &
described as being acid fast bacilli ( AFB ).

11. ( 1 ) Auramine phenol fluorescence stain
1 ) Staining the slide with auramine phenol for 10 min.
2 ) Washing with sterile water .
3 ) Adding 1% of acid alcohol for 5 min. & washing again with sterile water .
4 ) The slide then counterstained with 0.1 % potassium permanganate for 30 sec.
the TB bacilli are stained yellow against dark background & easily visualized using
florescent microscope.

13. Advantage :
- More sensitive
- Rapid
Disadvantages:
- Hazards of dye toxicity
- more expensive
- must be confirmed by Z-N stain

14. ( 2 ) Ziehl-Neelsen stain ( ZN stain )
1 ) Adding to slide kinyoun carbol fuchsin for 10 min.( which is a high
concentration of phenol in it & it does not require heat en as you need to do for
regular ZN stain).
2 ) Wash wih sterile water then add 3% of acid alcohol for 5 min.
3 ) Then counterstained with malachite green or methylene blue for 30 sec.

15. The sputum is stained by Ziel Nelseen stain and appear under
microscope as red bacilli

16. Advantage: - cheap – rapid
- Easy to perform
- High predictive value > 90%
- Specificity of 98%
Disadvantages:
o- sputum ( need to contain 5000-10000 AFB/ ml.)
oLow sensitivity; Reported sensitivity ranging 25 to
o65% when compared to culture
o- Young children, elderly & HIV infected persons may
onot produce cavities & sputum containing AFB.
o
o-Species differentiation impossible. False positive;

17. 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

18. B ) Culture on media
Divided into :-
1 ) liquid culture media: know as Bactec Mycobacterial Growth Indicator Tube
System(MGITS).
Tube contains modified Middle brook 7H9 broth base with OADC enrichment &
PANTA antibiotic mixture.

19. The PANTA antibiotic mixture
The OADC supplement
O ----- Oleic acid ( Metabolic stimulant)
A ----- Albumin ( to bind toxic free fatty acid )
D ---- Dextrose (Energy source )
C ----- Catalase ( Destroy toxic peroxides that may be present in the medium )
P ---- Polymyxin B
A ---- Amphotericin B
N ---- Nalidixic acid
T ---- Trimethoprim
A ---- Azlocillin
The antibiotic mixture inhibits the growth of contaminating bacteria.

20. BACTEC 460 ( rapid radiometric culture system )
Specimens are cultured in a liquid medium (Middle brook 7H9
broth base )containing C14 – labelled palmitic acid & PANTA
antibiotic mixture.
Growing mycobacteria utilize the acid, releasing radioactive
CO2 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.

21. Principle of the procedure:
A fluorescent compound (which is sensitive to
O2) 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.

22. ( 2 ) solid culture media
Lowenstein –Jensen medium is an egg based media with addition of salts, 5
% glycerol, Malachite green & penicillin
Because of its slow growth, it takes 4-6 weeks before small buff- coloured
colonies are visible on the medium

23. Advantages: - Specificity about 99 %
- More sensitive (need lower no. of bacilli
10-100 / ml)
- Can differentiate between TB complex &
NTM using biochemical reactions
- Sensitivity tests for antituberculous drugs
( St, INH, Rif., E)
Disadvantages: Slowly growing ( up to 8 weeks )

24. Another test is
Tuberculin skin test ( PPD )
Diagnostic tool for pre symptomatic tuberclosis.
–Take pieces of cell and inoculate under the skin
–If PPD is negative you are happy
–If PPD is positive you take a chest x-ray in which they look
for infiltration (cloudy lungs)

25. TST
• if erythema & induration follow after 48-72 hrs, the
test is positive
• TST is mediated by sensitized CD4 T-helper cells.
A positive Mantoux test indicates an area of induration of 10
mm or more in diameter
Negative chest x-ray means you take 6 months of
antibiotics
Positive means you take 2 years of anti TB drugs. If
You don’t take the meds you can be arrested.

26. blood tests
• New blood tests examine immune response to TB
antigens
• Draw blood from patient, expose white cells to TB
antigens, look for signs of immune response
• Two most common methods are ELISA and ELISPOT

27. 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.