1. Laboratory Diagnosis of
TB
• Presenter:
WATI VUKIALAU-VULAVA
LT/NTP - NORTH

2. Objectives
1.
2.
3.
4.
5.
6.
7.
History of TB
elements of DOTS
What the bacteria looks like
Lab component of diagnosing TB
Sputum collection procedures
What good and poor sputum quality is
Available Lab tests in Fiji to detect TB

3. History of TB
• TB has affected humans
for millennia
• Historically known by a
variety of names,
including:
– Consumption
– Wasting disease
– White plague
• TB was a death sentence
for many

4. Cont…History of TB
Scientific Discoveries in 1800s
• Until mid-1800s, many
believed TB was
hereditary
• 1865 Jean AntoineVillemin proved TB was
contagious
• 1882 Robert Koch
discovered M.
tuberculosis, the
bacterium that causes TB
Mycobacterium tuberculosis
Image credit: Janice Haney Carr

5. Elements of DOTS Strategy
• Political commitment to TB control
• Case detection by sputum smear microscopy
• Regular, uninterrupted supply of high quality
anti-TB drugs
• Directly Observed Treatment (DOT)
• Standardised recording and reporting

6. Mycobacterium tuberculosis
* long, slender, straight or curved, acid fast bacilli
* slow growers, obligate
aerobes, intracellular
bacterium
* structure composed of high
molecular weight acidic
waxes, mycolic acid, cord
factor

7. WHY DO WE COLLECT SPUTUM?
1. To detect & diagnose TB (mycobacterium TB)

8. •Smear-positive patients are
4-20 times more infectious
•Untreated, a smear-positive
patient may infect 10-15
persons/year
•Smear-positive patients are
much more likely to die if
untreated

9. 2. To monitor effectiveness of treatment

10. When is the best time??
• The best time, but not the only time to
collect a sputum specimen is upon
awakening in the morning
• When more than 1 specimen is required they
will be collected on different days
• Any specimens collected in one day will
count as the first (1) specimen

11. Sputum Collection
• 3 specimens considered

12. Cumulative Positivity
3 sputum samples are optimal
93%
100%
100%
81%
50%
0%
First
Second
Third

13. These data have helped form the basis of the
current three-specimen requirement for ruling out
pulmonary TB. The probable rationale behind this
recommendation is that some patients shed
mycobacteria irregularly and in small numbers and,
thus, increasing the number of specimens would
increase the yield. With regard to the
recommendation for early morning specimens, the
assumption is that M. tuberculosis would be present
in maximum concentration in sputum after pooling
overnight in the respiratory tract.

14. Specimen collection timing
• Spot-Morning-Spot WHO/IUATLD
recommendation
• Spot - 1st visit to the clinic
• Early morning- first sputum in the morning
• Spot - 2nd visit to the clinic

15. Instructions to Patients
Give clear instructions to patients on how to
collect the sputum:
Importance of sputum
examination for TB diagnosis
Need for real sputum, not
saliva
How to produce good sputum
How to open and close the
container
How to avoid
contamination of outside

16. Patient education:
the best specimen comes from the lungs

17. • Sputum should be collected under direct
observation, at least for the first time. This is
to insure that the patient is being properly
coached and is giving a good coughing
effort, as well as insuring that uncooperative
patients are producing their own sputum for
examination.

18. INSTRUCTION FOR SPUTUM
COLLECTION
1. Specimen collection should be done without the client rinsing
the mouth or brushing teeth
2. If possible, go outside or open a window before collecting the
sputum sample. This helps protect other people from TB
germs when they cough.
3. The cup is very clean. Don’t open it until you are ready to use
it.
4. Inhale 2-3 times, breathe out hard each time
5. Cough deeply from the chest
6. Place the container close to the mouth to collect the specimen

19. CON’T
7. Spit the sputum into the plastic cup. Avoid contaminating
the inside of the container and lid
8. Keep doing this until the sputum reaches the 5 ml line (or
more) on the plastic cup. This is about 1 teaspoon of
sputum.
9. Screw the cap on tightly so it doesn’t leak.
10. Write on the specimen bottle the patient’s name, hosp
number, the number of sputum collected, the date of
collection
11. Put the cup into the specimen bag and into a specimen
box
12. Submit this to the laboratory ASAP

20. • If the patient cannot cough up sputum,
advice him/her to try breathing steam from a
hot shower or a pan of boiling water.

22. Packing/Transportation Specimens
 Tightly cap labeled sputum bottle
 Cushion a box or carton with
cellulose(optional)
 Pack 3 specimens (well labeled) of same
patients in 1 biohazard specimen bag
 Place bag of specimen upright in the
prepared box

23. Packing specimens
Put several layers of absorbent paper in the bottom of the shipper.
Keep upright
Absorbent paper

24. Packing/Transportation Specimens
• Place the request form in the pocket of the
plastic
• Prepare a checklist and place it in a separate
bag
• Seal and label the box of specimen
• Send to nearest Health facility/hospital or
laboratory.

25. Sputum sample
What is a good sample?
• 3-5 ml
• Usually thick and mucous, but may be fluid with
pieces of purulent material
• Color varies from opaque white to green, reddish to
brown when blood is present
• Clear saliva is not suitable; but examine saliva if a
better specimen can not be produced, especially
for follow-up examinations
• A specimen mainly containing blood should not be
examined; patient should see a doctor for
immediate management

26. Best use of Collection Container
• A new container per patient
• To obtain a good quality sample, several
attempts by patient may be necessary.
• Use a new container on each attempt
• Patient to open container only when about
to collect specimen
• Container once opened should be
considered used (dispose accordingly)

27. Best container??
•
•
•
•
•
•
Clean
Plastic
Transparent
Wide mouth
Good seal
Easily labelled

28. Good Quality Sputum
Good quality sputum( mucoid)
Good quality sputum(purulent)
Blood stained

29. Poor sputum
quality
saliva or nasal secretions
are unsatisfactory. Saliva
from mouth is water and
thin.
Good sputum
quality
Sputum from lungs is usually
thick and sticky

30. Where can you store the sputum?
• You can store the cup in the refrigerator
overnight if necessary. Do not put it in the
freezer or leave it at room temperature.

31. Diagnosis

32. In Fiji, TB is diagnosed using the following
Lab methods
1) smear microscopy – to look for the
bacteria (AFB) microscopically
2) Sputum culture - to grow the
bacteria Mycobacterium
tuberculosis
3) GeneXpert MTB/RIF - cartridgebased, automated that can
identify Mycobacterium
tuberculosis(MTB) and resistance
to Rifampicin(RIF) within 2hr
period

33. 1. Direct Microscopy
M. tuberculosis is a acid–fast
bacterium, rod-shaped bacterium
measuring 2-4 x 0.2-0.5 μm. They
appear as bright red rods against a
contrasting background.
The Ziehl-Neelsen stain is used to
demonstrate the presence of the
bacilli in a smear. The technique is
simple, inexpensive and detects those
cases of tuberculosis who are
infectious.
M. tuberculosis appearing as bright red
bacilli (rods) in a sputum smear stained
with the Ziehl-Neelsen stain

34. AFB MICROSCOPY
Advantages
-Rapid
- High specificity (AFB in sputum = TB)
• All mycobacterium are acid fast, no exception ;
• > 98% for AFB in high burden countries
- Accurate diagnoses
- Using simple and available equipment
Disadvantage
Low sensitivity;
culture
Reported sensitivity ranging 25 to 65% when compared to
Species differentiation impossible. False positive;
mycobacteria.
Saprophytic

35. Reporting on AFB Microscopy
Number of bacilli seen
Result reported
None per 100 oil immersion fields
Negative
1-9 per 100 oil immersion fields
Scanty, report
exact number
10-99 per 100 oil immersion fields
1+
1-10 per oil immersion field
2+
> 10 per oil immersion field
3+

36. 3 smears = sensitivity of 1
culture
About 95% of infectious cases

40. 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
- Easy to perform
- High predictive value > 90%
- Specificity of 98%
Disadvantages:
- sputum ( need to contain 5000-10000 AFB/ ml.)
- Young children, elderly & HIV infected persons may
not produce cavities & sputum containing AFB.

41. Importance of AFB microscopy
• Sputum smear microscopy - the most
efficient tools of case finding in a
national tuberculosis control program
because of its ability to identify and
distinguish the cases with highest
priority in tuberculosis control.
• It is dependable if sputum specimens
are of GOOD QUALITY.

42. AFB smear-microscopy
Acid-fast bacilli (AFB) (shown in red) are tubercle bacilli

43. Acid fast smear showing TB bacilli

44. 2. TB cultures

45. demiology 3
Culture
M. tuberculosis grows in
Lowenstein Jensen medium
or Ogawa medium, which
contains inhibitors to keep
contaminants
from
outgrowing the organism.
Because of its slow growth, it
takes 6-8 weeks before small
buff-coloured colonies are
visible on the medium.
Typical small, buff coloured colonies of M.
tuberculosis on Lowenstein Jensen
medium

46. Identification from solid media
• Rate of growth: visible isolated
colonies in 2–4 weeks.
• Colony morphology:
– buff-coloured (never pigmented)
– rough
– waxy
– appearance of bread crumbs or
cauliflower.
From colonies , ZN
staining should be
performed
46

47. M. tuberculosis colonies
47

48. 3. GeneXpert MTB/RIF Test

49. TEST CRITERIA
Xpert MTB/RIF test should be done on;
1.Symptomatic patients with AFB negative (negative on 3
specimens)
2.Relapses suspect cases
3.HIV patients
4.Cases from high burden (MDR) countries
OTHER REQUIREMENTS
•Request forms for TB testing should be used
•Clinicians should complete request form with clinical diagnosis
•Specimen collected should be of good quality
•Lab Technicians should only do GeneXpert test if diagnosis falls on
any of the criteria above.
1.AFB positive not converting at 3 months of treatment

50. How could GeneXpert help?
• Low sputum smear positivity of PTB in HIV
patient is common due to:
– Poor immunity to localize the infection
– High possibility for TB dissemination to other organs
GeneXpert can detect more TB among
PLHIV

51. GeneXpert - Facts and
Background
•
•
Endorsed by WHO : 8 DEC 2010
18 months of rigorous assessment
of its field effectiveness in:
Early diagnosis of TB, as well as MDRTB
TB complicated by HIV infection, which
are more difficult to diagnose
•
The test could revolutionize TB
care!

52. How does the test work?
• Detects DNA sequences specific for Mycobacterium
Tuberculosis and Rifampicin resistance by PCR
• Based on Nucleic Acid Amplification Test (NAAT). The
Xpert® MTB/RIF
–
–
–
–
purifies
concentrates
amplifies (by real-time PCR) and
identifies targeted nucleic acid sequences in the
Mycobacterium tuberculosis genome,

53. TB point of care testing
• Simple
• Minimum 3 steps for sample
preparation
• Rapid
• 2 hours result availability
• Accurate
• Sensitivity : adult PTB
Smear+Culture+ = 95%
• Smear- Culture+ = 80%
• Specificity : adult PTB = 95%

54. GeneXpert test is available in Fiji
• 3 GeneXpert diagnosis sites in the microscopic
centres of National TB Programm supported by
WHO/ Global Fund Grant, Fiji: Labasa Hospital,
Lautoka and PJ Twomey Hospital.
• Testing done for:
Sputum Smear suspects case
MDR suspect cases and
PLHIV

55. Take-home messages
• Sputum smear microscopy - the most
efficient tools for detecting TB with highest
priority in TB control hence is dependable if
sputum specimens are of GOOD QUALITY.
• Sputum collection procedures needs to be
stregthened

56. THANK YOU
•Any burning questions????

57. SPUTUM SALIVARY DATA, 1st Qtr
HEALTH
FACILITY
TOTAL SPECIMENS
POOR SAMPLE QUALITY
DIAGNOSTIC /
FOLLOW UP
DIAGNOSTIC / FOLLOW UP
LABASA
155
58
SAVUSAVU
46
22
LEKUTU
2
0
WAINUNU
2
1
SEAQAQA
6
3
2
2
3
2
NABOUWALU
8
5
WAINIKORO
8
4
TAVEUNI
39
23
UNSPECIFIED
1
0
TOTAL
272
120
NAVAKAKA
N/STATION
DREKETI
% salivary
48

58. SPUTUM SALIVARY DATA, 2nd Qtr
HEALTH
FACILITY
TOTAL SPECIMENS
POOR SAMPLE QUALITY
DIAGNOS
TIC
FOLLOW UP
DIAGNOSTI
C
FOLLOW UP
LABASA
113
21
38
8
SAVUSAVU
39
2
12
0
LEKUTU
1
0
0
0
NADURI3
3
0
3
0
SEAQAQA
6
0
1
0
DREKETI
2
0
0
0
RABI
8
1
2
1
WAINIKORO
10
0
6
0
TAVEUNI
5
1
2
0
TOTAL
187
25
64
9
% Salivary
31