Transcript Laboratory Investigations

TB –
LABORATORY
INVESTIGATIONS
by
Dr. Zubaidah Abdul Wahab
Datin Dr. Ganeswrie Raj
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LEARNING OBJECTIVES
• To update current laboratory
investigations & techniques for
diagnosing TB
• To strengthen laboratory facilities for TB
management
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INTRODUCTION
• Diagnosis of TB is based on the detection
of acid fast bacilli (AFB) on smears &
culture of Mycobacterium tuberculosis
from clinical specimens.
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INTRODUCTION
• Specimen collection
• Lab investigations
– Microscopy
– Culture
– Identification
– AntiTB sensitivity
– Molecular
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SPUTUM COLLECTION
• Sputum1
• At least 2 specimens
• At least one early morning specimen
• For patients who are unable to spontaneously
expectorate adequate sputum specimens
• Sputum induction with nebulised hypertonic saline
• Fiberoptic bronchoscopy with bronchoalveolar lavage
• Gastric lavage especially in paediatric group
(neutralise with sodium bicarbonate)
1WHO,
2010
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OTHER SAMPLES
• Sterile body fluids
• include blood, CSF, pleural fluid, peritoneal
fluid
• should be collected aseptically
• transported immediately to laboratory in
sterile container
-
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MICROSCOPY
• Microscopy
•
•
•
•
Presumptive diagnosis
Sputum
Ziehl-Neelsen staining for AFB
Conventional microscope
• low sensitivity (20 - 60%)1
• Light emitting diode-based
fluorescence microscopy (LED FM)2
• 10% more sensitive
• shorter time spent
• quicker turnaround time
1Steingart KR
2Shenai
et al., Lancet Infect Dis, 2006
S et al., Int J Tuberc Lung Dis, 2011
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CONVENTIONAL LIGHT MICROSCOPY
Acid fast bailli
IMMUNOFLUORESCENCE MICROSCOPY
AFB bacilli – IF stain (low mycobacterial load)
CULTURE & SENSITIVITY
• Culture
• conventional method using LJ or Ogawa
• 6 - 8 weeks
• Drug-susceptibility testing
• 1 - 2 months
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PHENOTYPIC METHOD –
Proportion method
A
B
Susceptible
to INH
M. tuberculosis
Without drug
With drug - INH
Conventional culture medium (solid) –
TB. Rough, buff, cream-coloured
colonies, compact, corded colonies
CULTURE & SENSITIVITY
• Liquid culture media
a. Bactec MGIT
- detection by 2 weeks
- identification within 3 weeks
- sensitivity testing by 4 weeks
b. Bactec MycoF only for blood sample
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NUCLEIC ACID
AMPLIFICATION TEST (NAAT)
• NAAT
• molecular technique
• provide rapid results within 24 - 48 hours
• ability to confirm presence of Mycobacterium in 50 80% AFB smear negative & culture positive1,2
• detect Mycobacterium in specimens weeks earlier
than culture for 80 - 90% patients2
1CDC,
MMWR Morb Mortal Wkly Rep, 2009
2Sarmiento OL et al., J Clin Microbiol, 2003
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LINE PROBE ASSAY (LPA)
• Line Probe Assay (LPA)
• detect rifampicin & isoniazid resistance in
smear positive sputum or culture isolates
• carried out in a TB risk level 2 laboratory
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GENE XPERT MTB
• Gene Xpert MTB
• for detection of rifampicin resistance
Mycobacterium
• fast & accurate
• can be used in peripheral laboratories
• fully automated, near the patient, uses a
robust system & technically simple
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COMMERCIAL SEROLOGICAL ASSAY
• Recommendation 5
Commercial serological assay should not
be used to diagnose pulmonary &
extrapulmonary TB.
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COMMERCIAL SEROLOGICAL ASSAY
• WHO published a policy statement in
2011 stating that commercial serological
tests for TB provides inconsistent &
imprecise estimates of sensitivity &
specificity which can adversely impact
patient safety.1
1WHO,
Commercial serodiagnostic tests for diagnosis of tuberculosis, 2011
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ROLE OF ADENOSINE DEAMINASE (ADA)
IN TB DIAGNOSIS
• ADA is an enzyme involved in purine
metabolism of the cells.
• Measurement of ADA level in pleural effusion
& CSF is useful additional test in pleural &
meningeal TB.
• In pleural TB, sensitivity = 92% & specificity = 90%1
• In CSF-ADA in TB meningitis, sensitivity = 93% &
specificity of 97% if cut-off level of >10 U/L used2
1Liang QL
2Rana
et al., Respir Med, 2008
SV et al., Clin Neurol Neurosurg., 2010
TAKE HOME MESSAGES
• LED FM has more advantages than
conventional microscopy
• NAAT e.g. LPA is able to shorten the turnaround time for identification & antiTB testing
• Measurement of Adenosine Deaminase level
in pleural or cerebrospinal fluid may be
considered as an adjunct in diagnosing pleural
TB & tuberculous meningitis respectively.
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THANK YOU
[email protected]
[email protected]
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