LABORATORY DIAGNOSIS FOR

TUBERCULOSIS

Agus Sjahrurachman
Robert Koch on march 24th, . 1882
founded TB bacilli

March 24th is a TB day worldwide

INTERNATIONAL STANDARD CARE FOR TUBERCULOSIS ( 2009 )
ASPEK LAB
PIPELINE FOR LAB DIAGNOSIS OF TB
 POWER OF NEW DIAGNOSTICS

DETECTABLE MTB BY ZIEHL-NEELSEN STAINING MINIMUM 5000-10.000

BACILLI PER ML SPUTUM
 COMPARATIVE PERFORMANCE
DETECTION OF AFB IN SPUTUM

Xpert

•MGIT: 10-100 bacteria/ml sputum

•Detection limit for ZN: 10000 bacteria /ml sputum
•Detection limit for GeneXpert: 100-1000 bacteria/ml sputum
 AVAILABLE DIAGNOSIS METHODS

MICROSCOPY, direct and after cetrifugation at 3000g

CULTURE AND DST
SEROLOGY
INTERFERON DETECTION
PROBE AND NAAT
SKIN TEST
CHEST X RAY

MAIN DIAGNOSTIC TOOLS : AFB STAINING AND CULTURES.

HISTOPATOLOGI IS ADDED FOR EXTRA PULMONARY TB (
INTERNATIONAL STANDARD FOR TUBERCULOSIS CARE 2009 ).
MICROSCOPY
Diagnosis
Treatment follow up for NON MDRTB
Do not diffrentiate viable and dead bacilli
Do not differentiate species
Cut off value 5.000-10.000 bacilli per ml
Sensitivity 1 sputum 40-60%. 3sputum 80%
Lower sensitivity for HIV & pauci basiler Tb

ZIEHL NEELSEN (HOT)

MODIFIED-ZIEHL NEELSEN ( HOT )
KINYOUN ( COLD )
KINYOUN-GABBETT ( TAN THIAM HOK ) ( COLD )
AURAMINE FLUOROCHROME ( COLD )
 REPORTING ( IUATLD )

FINDING REPORT
0/100 lp -
1-9/100 lp Write number
10-99/100 lp +
1-10/lp,min 50 lp ++
>10/lp,min 20 lp +++

Observing 300 microscopic field is better than

observing 100 field. Up to additional 15% cases can
be detected
 USE OF MICROSCOPY IN TB CASE
MANAGEMENT

DOTS

MONTHLY MICROSCOPY IS USED FOR MDR-TB

TREATMENT FOLLOW UP
 CULTURE

Confirm the diagnosis

Treatment follow up for MDRTB and XDRTB
Speciation of mycobacteria
Isolate use for drug susceptibility testing
Cut off value 1000 bacilli per ml

NON SELECTIVE MEDIA :

LJ, ATS, MIDLDLEBROOK 7H10, MIDDLEBROOK
7H11,PETRAGANI
SELECTIVE MEDIA :
MODIFIED LJ, LJ PLUS, MIDDLEBROOK 7H10 PLUS,
MIDDLEBROOK 7H11 PLUS

LIQUID MEDIA PROVIDE SHORTER TURN AROUND TIME THAN SOLID MEDIA
 Mtb identification from isolate

Conventional biochemical reaction

Immunochromatographic test using specific MTB antigen
PCR-based method
INNOLIPA MYCOBACTERIA

Identify Mtb and impotant NTM

 EXISTING ANTIBODY DETECTION KITS

Not appropiate for diagnosis of adult

tuberculosis nor treatment follow up ( W.H.O
2008 )

Antibody responses varies : nutritional status, congenital and

acquired immune deficiencies, endemicity, etc
Specificity depend on the antigen used. Molecular mimicry is
not uncommon
 Celluler immune response detection
T-cells of individuals infected with M. Tuberculosis release INFY
when they re-encounter TB antigens OR even non specific
mitogens

QUANTIFERON : 1 st generation, similar to PPD at least 100

mycobacterial antigens ( shared with BCG and some NTM )
QUANTIFERON-GOLD : 2nd generation, use two mycobacterial
specific antigens: early secreted antigenic target 6 (ESAT-6)
and culture filtrate protein 10 (CFP10) .Not shared with the
BCG sub-strains and most NTM (except: M. kansasii, M. szulgai,
M. marinum and nonpathogenic M.bovis).
QuantiFERON –Gold : 3rd generation, use ESAT-6; CFP10; and TB7.7
(Rv2654) .
 INTERPRETATION of GOLD
QUANTIFRON

QUANTIFERON POSITIVE : MTB INFECTION LIKELY

QUANTIFERON NEGATIVE : MTB INFECTION IS NOT LIKELY
QUANTIFERON-TB GOLD TEST
FDA APPROVED
DOES NOT AFFECTED BY BCG
BASED ON IFNY PRODUCTION OF BLOOD CELLS AFTER
BEING EXPOSED TO ESAT6 AND CFP-10 ANTIGENS
MORE SPECIFIC THAN TST
DO NOT DIFFERENTIATED LTBI AND ACTIVE DISEASE
LIMITED DATA ON THE USE IN
in children younger than 17 years of age, pregnant
women persons recently exposed to Mtb,
immunocompromised persons (e.g., HIV nfection
or AIDS, current treatment with
immunosuppressive drugs, selected
hematological disorders, specific malignancies,
diabetes, silicosis, and chronic renal failure ( CDC
2009).
 ELISPOT assay (Oxford, UK)

Similar to QUANTIFERON TEST.

Measures number of reactive lymphocytes.

ELISPOT is no beter as compare to TST for culture-confirmed

paediatric cases, and even poorer compare to culture-
confirmed and clinically probable TB cases. ( Nicol,M.P, et al
2009 )
 ANTIGEN DETECTION

The detection of mycobacterial antigens by

immunoassay in clinical specimens with high &
variable protein content is difficult.

Detection in sputum presents even greater

clinical problem because sputum is a non-
homogenous gel .

False positive rates are high.

 NUCLEIC ACID AMPLIFICATION ( NAAT )

IN HOUSE NAAT IS NOT RECCOMMENDED

CAN BE USED FOR SPECIATION OF MYCOBACTERIA

 GENE TARGETS FOR AMPLIFICATION

65 Kd antigen (HSPs):
Genus-specific gene.
Unsuitable for detecting M.tb,particularly in areas where
species like M.avium or M.kansasii are prevalent.

IS6110 :
IS6110 found in the M.tb complex organisms (
M.tb, M.africanum, M.microti, M.bovis).
IS6110 sequence generally occurs only once in M.bovis
but is found as often as 20 times in certain strains of
M.tb,thus offering multiple targets for amplification. It is a
transposon which are self replicating stretches of DNA.

OTHER TARGET : 16S r RNA: genes encoding 38 kda, MPB64,mpt 40,

pmt64
COMMERCIALLY AVAILABLE STANDARD PCR BASED DIAGNOSTIC KIT

The Amplicor MTB Test

584 bp fragment of the 16S ribosomal RNA gene,
comprising a species-specific flanked by genus-
specific sequences, is amplified using biotinylated
primers.
Other Amplicor kits are available for detection of
Mycobacterium avium and Mycobacterium
intracellulare DNA in clinical samples.

Specificity is close to 100 % while sensitivity ranges

from 90 % to 100 % in smear-positive samples and
from 50 % to 95.9 % in smear negative ones
 TMA
A M. tuberculosis complex-specific region of the 16S ribosomal RNA gene
produces double-stranded ribosomal DNA. In turn, RNA polymerase
catalyzes the synthesis of multiple stretches of ribosomal RNA from the
ribosomal DNA synthesized before. The newly produced ribosomal RNA
undergoes further transcription by reverse transcriptase. The amplificons is
hybridized with a specific, chemiluminescent-DNA probe.

Amplified Mycobacterium tuberculosis Direct Test (AMTD, is a

commercially available isothermal (42°C) TMA System ,

Data from the huge amount of literature available

show sensitivity ranging from 91.7 % to 100 % in
smear-positive samples and from 65.5 % to 92.9 % in
acid fast bacilli (AFB) smear-negative samples .
 BD ProbeTec ET

Uses DNA polymerase and isothermal strand

displacement amplification to produce multiple
copies of IS6110

The literature reports a rate of sensitivity

ranging from 98.5 % to 100 % for smear positive
samples and very variable (0.33 %-100 %) for
smear-negative ones
 LAMP

Loop-mediated isothermal amplification.

It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity, efficiency, and rapidity.
LAMP is used for detection of M.tb complex, M.avium,and
M.intracellulare directly from sputum specimens as well as for
detection of culture isolates grown in a liquid medium (MGIT)
or a solid medium (Ogawa’s medium).
Species-specific primers were designed by
targeting the gyrB gene.

NOT YET RECOMMENDED BY W.H.O

 FDA APPROVED NAAT FOT MTB

Enhanced-Amplified Mycobacterium Direct Test ( E-MTD )

Sensivity for smear-positive cases >95%
Sensitivity for smear-negative cases 70-90%

Amplicor Mycobacterium Test

Sensitivity for smear-positive cases >95%
Sensitivity for smear-negative cases 60-70%

The positive predictive value of FDA-approved NAA

tests for TB is >95% in AFB smear-positive cases
when the clinical suspicion of TB is low, the positive
predictive value of the NAA test is <50%
 Application of NAA for TB in children and
extrapulmonary TB await further clarification

NAA does not replace the need for AFB smear

and culture as standard diagnostic
Mycolor TK and the diagrams obtained in the instrument. A) Typical growth curve of
a M. tuberculosis strain. B) The bar graph of the antibiotic susceptibility test of a M.
tuberculosis strain resistant to INH and streptomycin.

Not yet endorsed by W.H.O

Diagnostic Microbiology & Infectious Disease volume 72, 2012
 Magicplex

AB7500 COMPATIBLE CFX96™

Real-time PCR EQUIPMENTS Real-time PCR
Life FOR MAGIFLEX Bio-Rad
Technologies

Magicplex™ TB/MDR Real-time Test (V2.0) detects MTB and

screens for drug resistance to RIF and INH within additional 40
minutes. MDR-TB Real-time Detection employs multi-target
detection system screening for 7 major mutations causing
Rifampicin resistance and 3 major mutations causing Isoniazid
resistance.
 Anyplex™ MTB/NTM/DR-TB Real-time Test,
multiplex diagnostic test simultaneously
identifies tuberculosis, M avium complex-M
kansasii-M abcessus , multidrug-resistant TB
(MTB), and extensively drug-resistant TB.

Anyplex Plus screens for 5 mutations causing INH

resistance, 15 mutations causing RIF resistance, 9
mutations causing FQ resistance, and 6 mutations causing
injectable drug resistance.
 NEXT GENERATION............... POC DIAGNOSIS

hand-held molecular devices, breath-based assays for the

detection of volatile organic compounds, microchip
technologies and proteomics-based and metabolomics-
based tests
1. Instrument‐free sample preparation: Rapid
Extraction of Nucleic Acids

2. Isothermal nucleic acid amplification: Cross

Priming Amplification (CPA),

3. Visual read‐out detection and

easy data interpretation:
Rapid Nucleic Acid Detection
on Lateral‐flow Strips”
CURE THE REAL TB PATIENT

AVOID GARBAGE IN-GARBAGE OUT

IDENTIFY QUALITY LAB

BE AWARE OF MIS-DIAGNOSIS