Laboratory diagnosis of tuberculosis

Prepared by
Nahed Abedelateef
 Content Outline
• Introduction
• Specimen collection
• Safety measures
• Direct Smear Examination
• Fluorochrome stain
• T.B Culture
• Rapid tests for T.B diagnosis: Tuberculin skin test-
The MGIT 960 system-PCR
 Introduction

• Tuberculosis, also known as consumption, white

plague, and wasting disease, which has affected
humans for centuries.
• Until the 1800’s, it was widely considered to be a
hereditary disease.
• In 1882 the German scientist, Robert Koch,
discovered the bacteria responsible for causing
the disease, Mycobacterium tuberculosis.
 Purpose of of laboratory diagnosis of
T.B
• 1-Tuberculosis managers use the information to
ensure that bacteriologically proven patients are
receiving appropriate chemotherapy.
• 2-Health care workers use the diagnosis for
management of tuberculosis.
• 3-Public health authorities use them for statistical
and epidemiological purposes
 Specimen collection

• The advantages of Laboratory diagnosis of T.B

will not be fully realized unless specimens are
collected with the utmost care and promptly
transported to the laboratory.
 Common Sites of TB Disease
• Lungs
• Pleura
• Central nervous system
• Lymphatic system
• Genitourinary systems
• Bones and joints
• Disseminated (miliary TB)
• Pericardial fluids
 Sputum collection

• More than 85% of tuberculosis diseases in high

prevalence countries is pulmonary.
• Sputum is the specimen of choice in the
investigation of tuberculosis.
• If extra-pulmonary disease is suspected, sputum
should be collected in addition to any extra-
pulmonary specimens
 Patient Instructions: Sputum
Collection
Explain clearly to patient:
• Why sputum is needed
• Three samples required
– Spot-morning-spot
• What a “good” sample is and how to obtain it
• Opening and tight closing of containers
• Not to expose the sample to sunlight
• Transport of sputum containers
• The need to return to the clinic
 Patient Instructions: Sputum
Collection..
• Rinse mouth and throat with water two to three
times, and drink some water to wet throat (for easy
spitting of viscid sputum)

• Inhale deeply 2-3 times, breathe out hard each time

• Keep the body inclined to front
• Cough deeply from the chest
 Patient Instructions…

• Open the container and keep it near mouth

and spit sputum in Close lid securely
• Wash hands after handling sputum container
• Bring container to T.B laboratory.
Specimen collection
Labeling Specimen Container
Specimen collection
 Specimen Quality

Purulent Mucoid
 Specimen Quality

Saliva or Induced sputum (?) Blood stained

Poor quality sputum
 Sputum collection..

• When pulmonary TB is suspected, three sputum

specimens must be collected for examination
– Inpatient: three early morning specimens over
three consecutive days
 Timing of Specimen Collection
Spot–Morning–Spot
WHO/IUATLD Recommendation
Spot-early morning-spot specimen collection will detect
90% of smear-positive cases

• Spot initial visit to the clinic

• Early morning first sputum in the morning
• Spot second visit to the clinic
 Safety measures

• Laboratory workers are responsible for their own

safety and that of their co-workers.
• Infection control in the laboratory must aim at
reducing the production of aerosols.
 Mode of transmission

• Mycobacterium tuberculosis, is spread from

person to person via air.
• When an individual with infectious TB
disease coughs, sneezes, sings, or speaks,
minute particles containing M. tuberculosis
may be expelled into the air..
Mode of transmission…
 Personal Protective Equipment
• 1-Gloves
• 2-Laboratory Coats
• 3-Masks:N95
• 4-Appropriate Disinfectants
 Specimen Collection: Safety

• The patient is a greater danger to staff than the

specimen!
• Instruct patient to cover the mouth when coughing
• Never collect sputum in the laboratory!
– Collect OUTSIDE
– Collect away from other people
• Do not stand near patient during specimen collection
Advantages of Open Air Collection

• Rapidly dilutes aerosols

• UV light rapidly inactivates the
bacilli
 Obtaining adequate good
quality specimens is critical
to ensure accurate and
reliable AFB microscopy
results
 Specimen Collection: Safety

• Specimens should be received in the office area.

• Delivery boxes should be opened in the biosafety
cabinet .
• Inspect the delivery box for signs of leakage. If
mass leakage is evident discard the box by
autoclaving or burning.
Delivery boxes
 Specimen handling: safety cabinet

• All manipulations should be carried out within the

BSC.
• The cabinets are intended to protect the worker,
from airborne infection.
Class II
Laboratory arrangement
 Laboratory Arrangement

Microscopy Recording/Reporting
staining

Smear
prep
Reception of sputum
 First: Direct Smear Examination
 Advantages :
1. Rapid method of diagnosis .
2. Cheap.
3. Doesn’t need high skill & it is uncomplicated.
4. Gives a rough index for the number of bacilli
in the specimen.
5. Identify infectious patient.
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6. Follow up of patient under treatment.
 Direct Smear Examination

• Disadvantages :
– Less sensitive than the culture in diagnosis
of TB (a positive smear requires the presence
of about 5000-10000 acid-fast bacilli (AFB) per
ml of sputum.
– Less specific ( can’t differentiate between
typical & atypical mycobacteria ).
 Ziehl–Neelsen Method
I. Smearing :
 Enumeration: The slides are enumerated by the
diamond pencil. The slide number is written at the
edge of the slide.
 Smearing: Transfer the purulent part of the specimen
in the sputum container by wooden stick to the slide.
The precipitate of any sample is taken by Pasteur
‫سم‬2
pipette after sample centrifugation.
The sample is smeared by the wooden stick such that ‫سم‬3

it is homogenous thin and its area is 1 x 2 mm. The

sample is smeared in a spiral way as in the diagram.

34
 Ziehl–Neelsen Method ( Cont.)
N.B. : Don’t mix the specimen as this may lead to its
dilution.
 Drying: The slide is dried in air for 15 -30 minutes.
Never use the flame for drying. The smear shouldn’t be
exposed to direct sunlight, u.v. or high temperature
which may destroy the acid- fastness property of TB
bacilli.
 Fixation: The film is passed over bunsen flame 3 times
for 3 -5 seconds.

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 Ziehl–Neelsen Method ( Cont.)

2-Staining
Slides are put on the stand ( not more than 10-12) .A
space is left between each 2 slides.
 Cover the slides by filter papers , one for each slide.
 Add basic fuchsin on the slides.
 Heat gently by the flame till steam appears. Flaming is
done using cotton & alcohol. Avoid boiling or drying of
the stain . Heating is repeated 2- 3 times.

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 Ziehl–Neelsen Method ( Cont.)
 Leave the warm stain on the slides for 7 – 10
minutes.
 Wash each slide separately by the tap water after
removal of filter paper by a forceps.
 Re-put the slides on the holder.
 Decolrization with acid alcohol 3%.
 Flood the slides with the counter stain (methylene
blue) for 60 seconds.

 Wash each slide separately by the tap water.

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 Reading
A binocular microscope is recommended for
the examination of stained smear.
 Ensure that the condenser is in the upper
position with the diaphragm open.
 Place the slide on the mechanical stage & put
a drop of cider oil on the smear
 Examine the a slide with 100x immersion lens
so that it comes into contact with the oil.

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 Quality of Smear

Good
Too Thick

Sloughed off
Too Thin
Uneven
 Reading ( cont.)

40
 Microscopy report

Once completed, the microscopy report should be

made available as soon as possible, preferably no
longer than 24 hours after the laboratory receives
the specimen.
 Microscopy report..

• The microscopic observation must establish, first of

all, if there are acid fast bacilli present in the smear
• if positive the approximate average number of
these bacilli per microscopic field observed.
• It is recommended that a uniform pattern of reading
is followed, observing 100 useful fields.
 Microscopy report..

• A useful microscopic field is regarded as one in

which cellular elements of bronchial origin
(leucocytes, mucuous fibres and ciliated cells) are
observed.
 Negative
• Indicate the staining method.
• Report “negative for acid-fast bacilli” for all
smears in which no acid-fast bacilli have been
seen in 100 fields.
 Positive
• Indicate the staining method.
• Count the number of red bacilli per field.
• The number of acid-fast bacilli found is an
indication of the degree of infectivity of the
patient as well as the severity of tuberculosis
disease.
Quantification of Ziehl-Neelsen smear results
(IUATLD/WHO)
 The final microscopy report should contain the
following information:
• 1-evaluation of the quality of the sputum
specimen.
• 2-the staining method used: Ziehl-Neelsen smear
or fluorochrome staining method.
• 3-the average number of acid-fast bacilli seen on
the smear
• 4-indication of any large numbers of clumps,
which means that the actual number of acid-fast
bacilli may be larger than reported
 The final microscopy report should contain the
following information..

• 5-report only the number of acid-fast bacilli seen:

do not try to identify the mycobacterium species.
• 6-the date of examination and the microscopists
signature .
• 7-always send a report to the referring health
centre.
 The final microscopy report should contain the
following information..

• Never give the results only to the patient. If s/he

fails to bring the results to the health centre, s/he
may not receive the necessary treatment.
 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:
-More sensitive
-Rapid
Disadvantages:
- Hazards of dye toxicity
- More expensive
- Must be confirmed by Z-N stain
- Emission of UV is harmful to the eye
 3-TB Culture
Homogenization

• The definitive diagnosis of tuberculosis can only be made if

M. tuberculosis is isolated from the clinical specimen.
Advantages :
• Higher sensitivity than direct smear microscopy about 98% .
• Highly specific as you can identify the colony &differentiate
MTC from Mycobacteria other than tuberculosis or NMTC .
• Could identify MTC from different samples e.g pus,
pulmonary effusion, biopsy,C.S.F, urine.. etc.

56 TB Culture
 Disadvantage :

1. Turn over time to get the result ranges from 21 days to 8

weeks due to slow growth of MTC .
2. Digestion, decontamination & centrifugation of samples are
needed before culture.
3. Equipment needed are expensive .(BSC, autoclaves, ovens,
inspissations, incubators, water distiller) .
4. Contamination ranges between 3-4 %

57 TB Culture
 TB Culture...
Sample mixing

To obtain good results, we must kill the commensals in urine,

sputum, pus and throat swab samples by decontamination and
concentration.
Method of decontamination :
Petroff method ( Na OH method ) :
 Used in most samples especially in viscous ones as sputum and
pus because NaOH liquefies the organic substances and kills the
commensals .
 Don’t expose the samples to NaOH for > 45 min to avoid false
negative results because NaOH decreases the T.B Bacilli viability.
 TB Culture...
Phosphate buffer

- Procedure :
 Add equal amounts of sterile 4 % Na OH on the sample and close the bottle .
 Put in the shaker for 20 min .
 Centrifuge for 15 min (3000 RCF / min) .
 Neutralize the sediment by adding HCL ( 1N ) in phenol red for 15 min .
 Phenol red act as indicator for pH. Put the neutralizing agent drop by drop till
we reach point of neutralization which is the first drop that turn the sediment
color to yellow .
 Inoculate it on ( 2 media tubes of L.J. and 1 tube stonebrink media) using
Pasteur pipette (2-3 drops) .

59 TB Culture
 TB Culture...
 Put the inoculated media in a slanting position for 2-4
days in incubator to allow for good distribution of the
inoculum on The medium surface. Then put the
media vertically for 6-8 weeks at 35-37c .

Centrifugation
60
 Media of T.B. culture :
I. Solid media :
1-Egg based media LJ& Stone brink.
Growth in solid
Lowenstein Jensen media media
Slow: 15d-2m
Division time 18h
Stonebrink :
Used for isolation of bovine strains and INH-resistant
strains.
2.Agar based media:
( Middle- brook7H10 & middle – brook 7H11)
II. Fluid media :
Middle brook 7H-9 broth & 7H-12.
61 TB Culture
 Recording
• Accurate recordkeeping in the TB laboratory is essential.
• Recording means keeping the register up-to-date.
• Records should include information about the following
events:
• What type of specimens received by the laboratory.
• How specimen is identified.
• How results are reported.
• When specimens are sent to higher-level laboratories for
culture and drug susceptibility testing.
Rapid tests for T.B
diagnosis
 Tuberculin skin test

- It is performed by: intradermal injection ( Mantoux Test) of

0.1 ml of PPD ( Purified Protein Derivative).
- The test is read at 48 – 72 h .
- The reaction consists of erythema & induration.
- The diameter of the induration measured in milli meters.
- Induration of > 10 mm is virtually diagnostic
of past or present tuberculous infection.
 Interpretation

Induration > 15 mm:

Considered positive in all persons ( including those
who have had previous BCG vaccination)
 ‫‪Rapid tests for T.B‬‬

‫‪1- BACTEC 960‬‬

‫‪The MGIT 960 system is a non-radiometric‬‬
‫& ‪automated system that uses the MGIT media‬‬
‫‪sensors sensitive to O2 to detect the fluorescence.‬‬
‫مادة الفلوروكروم المشبعة باألكسجين وممزوجة بالسيلكون فى قاع‬
‫األنبوبة‪.‬عندما تنمو الميكوبكتريا‪ ,‬تمتص األكسجين الموجود‬
‫وتفرز ثانى أكسيد الكربون عندئذ ينشط الفلوروكروم ويشع‬
‫وتعطى األنابيب نتيجة ايجابية من ‪ 42-4‬يوم‪ .‬بعد ذلك تقرأ نتيجة‬
‫سلبية‪.‬‬
 BACTEC 960 ….

Advantages:
-The system holds 960 plastic tubes which are
continuously monitored.
- Early detection as the machine monitoring &
reading the tubes every hour.
Molecular Diagnosis of Mycobacteria

-Direct Detection of Mycobacteria from Specimens

(sputum)
-Identification of Mycobacterial Species from
Culture

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 PCR
• PCR is capable of detecting even 1-10 organism
in clinical specimen.
• Amplification leads to rise in nucleic acid to 106-7
copies in few hours (25-30 cycles)
• Results are available within hours rather than
weeks.

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 When should PCR not be used?
• Do not use if patient has taken TB medications in the past
12 months.
• Can detect nucleic acids from dead and live organisms, so
may remain positive long after treatment is completed and
the culture is negative.

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 Using PCR with specific primer

 Primers are single-stranded 18–30 b DNA fragments

complementary to sequences flanking the region to be
amplified.
 Primers determine the specificity of the PCR reaction.

5’-ACTCCGAAGAAGCCGA-3

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Mycobacterium tuberculosis H37Rv genome.

75
DNA EXTRACTION

76
PCR:
Polymerase
Chain Reaction

77
 Electrophoresis

 Detect DNA band by

electrophoresis
PCR detection of M. tuberculosis
Sensitivity -- 92
Specificity -- 100

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 Advantage of PCR
 Can be detected using PCR within 3-5 hours
while cultures take weeks.
 Save time and money on drugs, contact
investigation.
 Earlier diagnosis for smear -
 Faster diagnosis, decrease TB transmission
 It’s easy (no extra sputum needed)
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 Limitations of PCR

 False positive:
• Due to contamination, carry over from
previous test
• lab personnel, technical error
 False Negative:
• Improper sample collection/ preparation
• Presence of inhibitors to Taq polymerase
80
 Interpretation of Results
• Not a perfect test.
• Does not replace culture results which are the “gold
standard”.
• Interpret within the patient’s symptoms, chest x-ray,
smear and culture.

81
 Smear+, PCR+
• Presume active TB disease
• Start TB medication
• Keep in isolation until cleared
• Confirm by culture result

82
 Smear+, PCR -

• Suspect nontuberculous mycobacterium.

• Suspect of PCR inibition
• Does not rule out TB
• If highly suspected of TB or lives in congregate
setting or with high risk individuals request a
second PCR.
• Confirm findings with culture result
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 Smear -, PCR +

• Likely has active TB disease

• Consider submitting another specimen for PCR
to verify.
• Presumed to have TB if two or more specimens
are PCR positive
• Use clinical judgment to determine whether to
start treatment, and place on isolation.
• Confirm by culture result.
84
 Smear-, PCR-
• For smear- specimens, sensitivity is low
• Diagnosis of TB cannot be excluded
• Requesting a second PCR may be helpful
• Patient considered non-infectious if sputum
smear- x 2 and all PCR results are negative.
• Confirm by culture result.

85
 Inhibited PCR
• Amplification was inhibited due to a naturally
occurring inhibitor in the specimen or processing
reagent (example: blood).
• Can result in a false negative.
• lab will request additional specimen to repeat the
test.

86
 Dr. Adnan Al-Hindi, PhD. Medical
parasitology (Training course in the diagnosis
18 ،‫ آذار‬06 of parasites in nwater and food) 87