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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 et al., Lancet Infect Dis, 2006
2Shenai 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

M. tuberculosis Susceptible
to INH

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 et al., Respir Med, 2008
2Rana 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 turn-


around 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

dr_zubaidah@sel.moh.gov.my
ganiraj_balan@yahoo.com

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