MICROBIOLOGY OF TUBERCULOSIS

Dept Microbiology, FMUI

HISTORY
 History…..

Robert Koch on march 24th, .

1882 founded TB bacilli

March 24th is a TB day

worldwide
 TB IN INDONESIA
•Rank no 3 in term of disease burden globally
•Rank no 3 as major cause of death ( SKRT1995 )
•New cases 583.0000 annually
•75 % cases are in productive age

Control program has not yet reached optimal

Why ? conditions
Increasing HIV cases
Affects mainly poor and less educated people
 CLASSIFICATION

Always human patogens :

M tuberculosis
M bovis
M leprae

Potentially pathogen,common Potentially pathogens, Uncommon

cause : cause :
• M avium complex • M Africanum
• M kansasi • M genavenae
• M haemophyllum
Rarely cause disease : • M malmoense
M gordonae • M marinum
M flavesens • M scrofulaceum
M fallax • M simiae
M gastri • M ulcerans
M smegmatis • M fortuitum
M terraetriviae • M chelonae
 HABITAT
TYPE HABITAT LESSION
Tuberculosis compleks
M. tuberculosis Human Bronchopulmonal
M bovis Human & cattle Soft tissue & GI tract
Photochromogen
M kansasii Water & cattle Skeletal
M marinum Water & fish Skin & soft tissue
M simiae Primate Bronchopulmonal
M asiaticum Primate Pulmonal
Scotochromogen
M scrofulaceum Soil.water & food Lymphadenitis
M szulgai ? Bronchopulmonal
M gordonae Water Pulmonal
M flavescens Pulmonal Soil & water
M xenopi Water Bronchopulmonal
 Habitat…..
TYPE HABITAT LESSION
Nonphotochromogen
M avium complex Soil,water,bird, cattle, Pulmonal, lymphnode,
swine generalized
M ulcerans ? Skin & soft tissue
M gastrii Soil & water Pulmonal
M terrae Soil & water Pulmonal
Rapid grower
M fortuitum Soil,water,animals, Skin,soft tissue,generalized
marine life
M abscessus Soil,water,animals, Skin, soft tissue, generalized,
marine life skeletal
M chelonae Soil,water,animals, Skin, soft tissue, generalized,
marine life skeletal

M smegmatis Humid surface Pulmonal

M leprae Human ,armadillo Skin,soft tissue,generalized
 MYCOBACTERIUM
 Straight or slightly curved rods, 0.2~0.61.0~10 µm
coccobacillary, filamentous and branched also
 Gram-positive, acid-fast, non-motile, non-sporing
 Pigmented in dark or after exposure to light
 Aerobic or microaerophilic
 Subdivision: rapid growers, slow glowers
 Large amounts of lipid in cell wall: mycolic acid
 A mol % G+C or 61-71%
 Typical species:
 Mycobacterium tuberculosis
 Mycobacterium tuberculosis

 Straight or slightly curved rods, 2~40.3~0.5 µm

 Singly or in small clumps (in specimens), serpentine cords (in
broth)
 Gram-positive, difficult to stain
 Does not grow on ordinary culture media, but only on enriched
media egg-based, agar-based, broths with bovine serum
 Grows slowly, generation time of cells in best condition of
culture on solid media : 13-20 hours
Mycobacterium tuberculosis Complex

 M. tuberculosis
 M. bovis
 M. bovis BCG
 A caprine (goat) variant or M. bovis
 M. africanum
 M. microti
 M. canetti
 Mycobacterium tuberculosis Complex….

 Colonies: a buff color, dry breadcrum-like, heaped up and

luxiriant or “eugonic”
 Optimal growth temperature: 35-37C
 Obligate aerobes, improves growth in CO2 atmosphere
 Survive in milk and in other organic materials
 Very sensitive to UV, also-heat-sensitive
 Susceptible to alcohol, formaldehyde, glutaraldehyde
 Resistant to drying
 Resistant to many antibiotics due to :hydrophobicity of
cell wall,production of beta lactamase, aminoglycoside
acetyl ,transferase, drug efflux system
 Mycobacterium’ s cell wall
contains peptidoglycan and a
large amount of glycolipids,
especially mycolic acids.

The peptidoglycan layer is

linked to arabinogalactan
(D-arabinose and D-galactose)
which is then linked to high
molecular weight mycolic acids.

The arabinogalactan/mycolic
acid layer is overlaid with a
layer of polypeptides and
mycolic acids consisting of free
lipids, glycolipids,and
peptidoglycolipids.

Other glycolipids include

lipoarabinomannan and
Structure of an Acid-Fast Cell Wall phosphatidyinositol mannosides (PIM).
 Patogenesis of M. tuberculosis

 Multiply extracelluar, upper lung lobus as

preference site

 Dormant in macrophage as survival strategy of

M . tuberculosis in the infected host
 neutralize phagosomal pH  arrest of
phagosome-lysosome fusion, phagosomal
maturation  prevent killing and degradation of
M. tuberculosis. Resting macrophages have weak
antimicrobial capacities

 No response to anti TB drugs

 Transmission of Tuberculosis

1. Direct contact
2. Directly inhales droplets generated when
persons with pulmonary or laryngeal TB sneeze,
cough, speak or sing
3. Indirectly inhales droplet nuclei generated from
an infectious patient in small, enclosed room
4. Indirectly inhales dusts containing TB in floor,
cloths or quilts
 Factors Influencing
Transmission

 The source case

 The environment, including ventilation
 The duration and intensity of exposure
 The contact
 The tubercle bacillus itself
LABORATORY INVESTIGATION
FOR MYCOBACTERIOSIS

Microscopic
• Direct
• Concentrated
Cultur & dst
Antibody detection
Cytokine measurement
Molecular
• PCR
• LCR
• Hibridization
DIAGNOSIS OF MTB
 POWER OF NEW DIAGNOSTICS

DETECTABLE MTB BY ZIEHL-NEELSEN STAINING MINIMUM 5000-10.000

BACILLI PER 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 2008 ).
 SPUTUM SPECIMEN
( NATIONAL CONTROL PROGRAM )
• Collect spot-morning-spot sputum
• Select a good wide-mouthed sputum container, which is disposable, made of
clear thin plastic, unbreakable and leak proof material.
• Give the patient a sputum container with the laboratory serial No. written on
it. Show the patient how to open and close the container and explain the
importance of not rubbing off the number written on the side of the
container.
• Instruct the patient to inhale deeply 2-3 times, cough up deeply from the
chest and spit in the sputum container by bringing it closer to mouth.
• Make sure the sputum sample is of good quality. A good sputum sample is
thick, purulent and sufficient in amount (2-3 ml).
• Give the patient another container with laboratory serial number written on
it for an early morning specimen. Explain to the patient to rinse his/her
mouth with plain water before bringing up the sputum.

Induction : expectorants, light sport, nebulizer

Storage and transportation of specimens

If the specimen is collected in the field and cannot be

immediately processed, it should be transported to the
laboratory within 3-4 days of collection.

The specimen should be collected in the containers meant

for the purpose, lid tightly secured, properly labelled and
kept away from the sun and heat.

These can be placed in a special box which can withstand

leakage of contents, shocks and other conditions incident
to ordinary handling practices. These boxes should be kept
in the cooler conditions and then transported to the
laboratory.
 MICROSCOPY
Diagnosis
Treatment follow up , 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 )
 Sputum Smear-Positive Persons

 Expectorate 108-109 bacilli daily or 106-107 acid-

fast bacilli/mL of sputum
 “The most contagious patient”
 Usually present about half of all newly discovered
cases of tuberculosis
“The chief targets of TB control program”
 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
 Slide evaluation

1. Quality of specimen : macroscopic ( mucopurulent ),

microscopic ( no of epithels and leucocytes )
2. Dimension 2x 3 cm
3. Eveness
4. Thickness
5. Staining quality
6. Cleaness

Microscope Observer competency

Quality microscopic result

 Sputum specimen, not saliva
Dirty
stain

Inapropitae decolourozation

Good
stain

good staining

Too thin
2x 3 cm, even

apropiaate too thick Small and uneven

Too large, un even
 Consequences of false smear
negative results

 Patients with TB will not be treated, resulting in

suffering, spread of TB and death

 Intensive phase of treatment will not be extended for the

required duration, resulting in inadequate treatment

 Patient may lose confidence in the programme

 Consequences for false smear
positive results

 Patients are started on treatment unnecessarily

 Treatment is continued longer than necessary, in
follow-up examinations
 Medications will be wasted
 Patients lose confidence in the programme
 USE OF MICROSCOPY IN TB CASE
MANAGEMENT

DOTS

Monthly microscopy is used for pmdt follow up

 SPECIMEN

Process ASAP, some is contaminated by normal flora

Appropiate volume

Morning sputum and urine is better

Avoid swab

Type of specimen depend upon infection site

Educate patient on proper method of expectoration
Acid Fast and Auramine Staining Microscopy
Acid fast bacilli are approximately 1-10
m long, slender, rod-shaped bacilli
which may be curved or bent. These
may be granular, isolated, in pairs or in
groups. Stained bacilli may present a
beaded appearance.

Weaker AFB BACTERIA

Rhodococcus
Nocardia
 Sputum Smear Microscopy

 Relative simplicity allows it to be performed even

in places without electricity
 It yields an immediate result, provides actual
proof of the presence of bacilli which make it
possible to start effective treatment
 Less prone to misinterpretation and less
expensive than chest radiography
 Limitation of Sputum Smear
Microscopy

 Relatively insensitive compared with isolation

by culture

 Cannot distinguish between

M. tuberculosis and nontuberculous
mycobacteria nor dead and living bacteria
 Sensitivity and Specificity of
Sputum Smear Microscopy

 A single smear of a respiratory specimen has a

reported sensitivity of between 22 to 43%
 When multiple specimens are examined, the
detection rate improves to 65-96%
 Most published studies show the acid-fast smear
microscopy to continue to have excellent specificity
(>90%), and positive predictive value (91.5-98%)
for diagnosis of tuberculosis
 ISOLATION AND IDENTIFICATION

Confirm the diagnosis

Differentiate MTb and NTM ( MOTT )
To be follow by DST
Cut off value 1000 AFB per ml

Non selective media :

• LJ, ATS, Midldlebrook 7H10, Middlebrook 7H11, Petragani

Selective media :
• Modified LJ, LJ plus, Middlebrook 7H10 plus,
Middlebrook 7H11 plus
Mycobacteria IDENTIFICATION METHODS
Growth rate and temperatire
Morphology and colour of the colonies
AFB staining
Biochemical tests :
Niasin
Nitrate reduction
Catalase,urease,arylsulphatase activity
Inhibition by thiphene carboxyilate hydrazide
Hydrolisis of tween 80
Pyrazinamide susceptibility
Growth on 9 % NaCl
Iron up take
TCH
Inhibition by PNB
BACTEC NAP differentiation test
Molecular tests,eg PCR
GLC,HPLC
 CULTURE

 Mycobacterial culture is more sensitive and specific, but

it is costly, time consuming, and required specialized
safety laboratories

 Usually not performed in most low-income countries

 The growth of M. tuberculosis requires 3 to 8 weeks,

waiting for positive cultures prior to the initiation of
therapy may be undesirable and impractical ( shorter
time in broth method )

 Isolates can be subjected to best drug susceptibility

testing method
 STANDARDIZED Drug Susceptibility Test

Proportion method
Absolute/break point method

Egg-based media ( LJ ) vs agar-based-media

Solid vs liquid media

Monitoring MDR TB
Drug selection
 Antitubercular Drugs
 First line drugs
 Rifampicin
 Broad spectrum, bactericidal
 Mechanism of action :
 binding bacterial RNA polymerase  interferes RNA
synthesis
 Ethambutol
 Inhibits biosynthesis of arabinogalactan (major
polysaccharide of cell wall)
 Isoniazid
 Inhibits mycolic acid biosynthesis (activated by KatG gen)
 Streptomycin
 Aminoglycoside
 Mechanism of action
 Interferes protein synthesis, damage of cell membrane,
misreading/miscoding genetic code
AIMS OF TREATMENT

 1. To cure patients and render them non-infectious.

 2. To reduce morbidity and mortality
 3. To prevent relapse and emergence of resistant
tubercle bacilli
 FACTORS FAVOURING 0CCURENCE OF
RESISTANT Mtb

Poor compliance
Low quality drugs
Inappropiate drug regimens and treatment duration
Co-morbid :
Cellular Immunodeficiency
Disorder that interfer with pharmacokinetic and
pharmacodynamic of drugs
 RELATED GENE FOR RESISTANCE TO ANTI TB
DRUG GENE FUNCTION ROLE TYPE#

H katG Catalase-peroxidase Prodrug conversion R

inhA Enoyl ACP reductase Drug target D
Nadh NADH dehydrogenase Modulator of INH activity R
ahpC Alkyl hydroperoxidase Marker of resistance
R rpoB RNA polymerase Drug target D

Z pncA Nicotinamidase Prodrug conversion R

E embCAB - Drug target D

S rpsl, S12 ribosomal protein Drug target R

rrs 16s rRNA D
A/K rrs 16s rRNA Drug target D

Q gyrA DNA gyrase A Drug target D

gyrB DNA gyrase B Participate in drug binding

lfrA Efflux protein

Et etaA/ethA Flavinmonooxidase Prodrug conversion R
inhA - Drug target

# type of allele
 MECHANISM OF RESISTANCE

Drug Genes Notes

H katG, 50-90% in katG, 20-35 in inhA
inhA,Nadh.ahpC gene,10-15% in ahpC-oxyR
gene. Mutation at other sites are
rare

R rpoB >95%) of R-resistant Mtb is due

to mutation at 81 base pair
region ( codon 507-533 ).
Resistance might also be caused
by mutation at gene resposible
for drug transport
 Mechanism of resistance to ethambutol (EMB )

EMB interacts with the EmbCAB proteins encoded by the embC, embA, and embB
genes, leading to inactivation of arabinogalactan synthesis.

Mutations in the embB locus cause alterations in EmbB, possibly leading to an altered
target for EMB. Alternatively, hyperexpression of the EmbCAB proteins could lead to
EMB resistance.

Inlet box: Organization of the emb operon in Mycobacterium tuberculosis (MTB).

 Mechanism of resistance to ethambutol (EMB )….

Resistance to FLQs, AM-CM in M. tuberculosis is most frequently

attributed to mutations in the gyrA and rrs ( 16S rRNA ), respectively.

70-90 % of FLQ-resistant strain is due to mutations in codons 90, 91,

and 94 in the gyrA gene.

Mutations at A1401G, C1402T, and G1484T in the rrs gene confer

resistance to CM, CM, and KAN , each of them being responsible for a
specific resistance pattern.

Mutations G1484T and A1401G were found to cause high-level

resistance to all drugs, whereas C1402T causes resistance to only CM
and KAN. Mutation of the tlyA gene, encoding a putative rRNA
methyltransferase, confers capreomycin and viomycin resistance
 TYPE OF RESISTANCE
Mono resistant
Poly resistant
Multi Drug Resistant
Pre extremely drug resistant
Extremely Drug Resistant
Pan Resistant
: 1 drug
: > 1 drug, not MDR
: at least R and H
: MDR plus 1
fluoroquinolones or 1
injectable drug
: MDR plus 1
fluroquinolones and 1
injectable drugs
: all drug
AMPLIFICATION OF DRTB

SPONTANEOUS SELECTION BY
MUTATION ANTI TB DRUGS
 OPTIONS TO DETECT DR TB

PHENOTYPIC TEST
Standardized methods for R,H,E,S, some second line is available
Time to reportable result 2-6weeks and beyond
Requires laboratory level of biosafety
Complex procedures
Confirmatory test

GENOTYPIC/MOLECULAR TEST
W.H.O endorsed methods is available
Turn around time is faster
Lower level of laboratory biosafety
Available for R,H,E, fluoroquinolones and aminoglycoside
Not suitable for treatment follow up
 PHENOTYPIC DRUG SUSCEPTIBILITY TESTS FOR MTB
PHENOTYPIC TESTING
• Solid-based media : Lowenstein-Jensen; Ogawa, TK media
Liquid-based media : Middlebrook,7H11,7H10

• Absolute drug concentration method

Proportion method

• W.H.O endorsed method

Proportion method on LJ or Ogawa
Absolute concentration method on liquid media : i.e Nitrate
reductase test ( NRA ), Colorimetric method, Microscopic
Observation Drug susceptibility ( MODS )
No validated methods for majority of second line
drugs, except for fluoroquinololnes and
aminoglycosides

Pyrazinamide testing can only be done in liquid

media

Drug susceptibility tests for mycobacteria other

than tuberculosis (mott )/non-tuberculosis
mycobacteria (ntm) Use different methods.

Only few drugs are aplicable

 MOLECULAR SUSCEPTIBILITY TESTS
FOR MTB

Hybridization of probes on isolates DNA

Amplification of selected gene fragments follow by detection of

amplicon on agarose

Amplification of selected gene fragments follow by hybridization of

amplicons with selected labeled-probe

Automated amplification of selected gene fragments through real

time polymerase chain reaction

Validated for rifampicin and isoniazide

 W.H.O ENDORSED MOLECULER DRUG
SUSCEPTIBILITY TESTS

Line probe assays :

Amplification of certain gene fragment follow by
hybridization with selected probes. MDRTB plus ( R
and H ), MDRTBSL ( OFLOXACIN, KANAMYCIN,
ETHAMBUTOL), INNO LIPA rif TB ( RIFAMPICIN )

Genexpert :
automated real time
Polymerase reaction ( rifampicin )

the proportion of rifampicin (RMP)

resistant isolates that were isoniazid (INH)
susceptible by phenotypic drug
susceptibility testing varied widely (0.5-
11.6%) W.H.O 2012.
 Placement plan of GeneXpert

Nort Sumatera

1. Adam Malik Hosp

DKI Jakarta Papua

East Java 1. BLK Papua

West Java Bali

1. Microbiology – UI South Sulawesi
2. Persahabatan Hosp
3. Pengoyoman Hosp. (Prison) 1. Sanglah Hosp.
1. Labuang Baji Hosp.
1. Hasan sadikin Hosp. 1. Soetomo Hosp. 2. NEHCRI Makassar
2. BLK Bandung 2. BBLK Surabaya
c 3. Saiful Anwar Hosp.
Central Java
DIY
1. Moewardi Hosp.
2. Kariadi Hosp.
1. Microbiology UGM Red color indicate W.H.O
3. Cilacap Hosp. (Prison)
certified labs for phenotypic dst
 MTBDR by HAIN Lifescience LiPA RIF.TB by
INNOGENETICS
Conjugate ctrl marker line
Universal ctrl
MTBC MTBC
rpoB universal ctrl

rpoB wild-type, 5
segments

516
526 4 rpoB mutations
531
katG universal ctrl

katG wild-type
315 2 katG mutations

5
9 More probes are added in MTBDRsl to detected 2nd-line drug R-mutations.
 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
 SEROLOGY

Not appropiate for diagnosis of adult tuberculosis

nor treatment follow up

Antibody responses varies : nutritional status, congenital

and acquired immune deficiencies, endemicity, etc
Specificity depend on the antigen used. Molecular mimicry
is not uncommon
 EVALUATION OF SEROLOGY TEST
( WHO 2008 )

Product of 19 companies :
Mycodots 9 Easy Step, TB Rapid Screen Test, TB-STAT PAK II,
Immune Sure TB Plus, SD TB Tapid test, TB-Spot Ver 2.0, TB
Rapid test, dBest One Step TB Test, BIOLINE Tuberculosis test,
etc

Specimen :
TB(+)HIV(+); TB(+)HIV(-);TB(-)HIV(+);TB(-)HIV(-). N 355
Collected from TB non endemic and endemic countries

Analysis :
Specicity, Sensitivity, Reproducibility, etc
 Evaluation of serology test
( who 2008 )

Overall sensitivity ranged from 1% to 60% and was higher in

sputum smear positive than sputum smear negative and
amongst hiv positive samples.

The majority of products had poor specificity ( <80% ) when

tested in tb suspects from endemic setting. Test with specificity
over 90% detected less than 30% of all tb patients

Hiv co-infection might diminishes the performance of the assays

Some products show high lot to lot, run to run, operator to

operator and inter-reader reproducibility
 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

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-BASED
DIAGNOSIS ( 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 on a solid medium (Ogawa’s medium).

Species-specific primers were designed by targeting the

gyrB gene.
 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%
 Approved NAA is less sensitive than culture. Negative
results is thus does not eliminate a possibility of having
TB. NAA tests can reliably detect ( in 80-90 % cases )
Mycobacterium tuberculosis bacteria in specimens 1 or
more weeks earlier than culture

Sputum specimen may contains NAA inhibitors ( up to

20% ) that may decrease or lead to false negative result,
although inhibitors does not cause false negative NAA
result in smear positive cases ( < 3 % )
Currently available –FDA approved NAA tests are not sufficiently
sensitive (detecting 50%--80% of AFB smear-negative, culture-
positive pulmonary TB cases) to exclude the diagnosis of TB in
AFB smear-negative patients suspected to have TB .

NAA results often remain positive after culture results

become negative during therapy.

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