Tb newer diagnostics

Dr. Namrata Kewalramani
Tuberculosis
newer diagnostic methods
Dr.T.V.Rao MD 1

Need for newer diagnostic methods??
 Early diagnosis of Tuberculosis and optimal treatment
- ensures cure of an individual patient
- curbs the transmission of disease in the community
 Case finding remains the corner stone for
effective control among distinct components
of TB control programme

• LED Microscopy
• Liquid Culture and DST
• Non commercial culture and DST (MODS, NRA, CRI)
• NAAT (PCR, LPA, Xpert MTB/RIF)
• Serological Antibody detection test
• Antigen detection Test – Urinary Lipoarabinomannan
(LAM)
• Point of care (POC) test
• IGRA

WHO recommendations on sputum smear microscopy
(2010)
• ZN light microscopy performed on
UNCONCENTRATED sputum is suitable for all
laboratory service levels
• Concentration of sputum is NOT recommended in
programmatic settings
• Fluorescence microscopy is recommended for
increased sensitivity (add 10%)
• LED microscopy is recommended over
conventional fluorescence
5

Fluorescent light-emitting diode (LED)
microscopy
• Conventional fluorescence microscopy is limited by
1) high cost of mercury vapour light sources,
2) the need for regular maintenance
3) requirement for a dark room.
• Light-emitting diodes (LEDs) are
1) less expensive light source
2) attachments require less power and can run on batteries,
3) bulbs have a long half-life and do not release potentially toxic
products if broken.
• The diagnostic accuracy of LED microscopy was found to be
comparable to that of conventional fluorescence microscopy and
superior to that of conventional Ziehl-Neelsen microscopy.

Liquid Culture and DST
• Egg-based media, such as Lowenstein-Jensen (LJ) or Ogawa
have been used for cultivation of mycobacteria for several
decades.
• In 1958, Middlebrook and Cohn described an agar based
medium to permit more rapid detection of mycobacterial
growth.
• However, it still required an average of 3-4 weeks to
recover mycobacteria from clinical specimens.
• In 1969, Deland and Wagner developed a technique for
semi-automated detection of the metabolism of bacteria by
measuring the 14CO2 liberated during the growth and
decarboxylation of 14C-labeled substrate incorporated in
the growth medium. This radiometric technique was widely
used for blood culture using the BACTEC 460 instrument.

• In 1980, this technique was introduced commercially
for mycobacterial recovery from clinical specimens and
drug susceptibility testing.
• The BACTEC 460 TB System has been reported to yield
15-20% increased culture positivity with an average
time-to-detection of positive growth from 8 to 14 days
as compared to 3 to 5 weeks on solid media.
• The high efficiency of the BACTEC TB System is due to
the use of liquid medium.
• Moreover, a growth enhancing substance is added to
the medium to further reduce the detection time.

• Disadvantages of the BACTEC 460 TB System
is the use of 14C-Labeled radioactive substrate.
Because of the strict regulations of handling
and waste disposal of radioactive material, it
became necessary to develop a non-
radiometric technique for mycobacterial
culture and susceptibility testing.

• Becton, Dickinson and Company (BD)
developed a new system called Mycobacteria
Growth Indicator Tube (MGIT™)
• Its is non-radiometric and offers the same
rapid, sensitive and reliable methods of
testing
• It is the fully automatic system for detection
of mycobacterial growth and drug
susceptibility testing of M. tuberculosis

MGIT medium
• consists of liquid broth medium th MGIT
• 7.0 ml of modified Middlebrook 7H9 broth base
• medium is terminally sterilized by autoclaving.
• enrichment
- MGIT OADC (Oleic acid, Albumin, Dextrose and
Catalase)
-MGIT 960 Growth Supplement, is added
• Growth Supplement is essential for growth of
many mycobacteria, especially those belonging to
M. tuberculosis complex.

Principle
• MGIT tube contains an oxygen-quenched fluorochrome, tris 4, 7-
diphenyl-1, 10-phenonthroline ruthenium chloride pentahydrate,
embedded in silicone at the bottom of the tube.
• During bacterial growth within the tube, the free oxygen is utilized and
is replaced with carbon dioxide. With depletion of free oxygen, the
fluorochrome is no longer inhibited, resulting in fluorescence within the
MGIT tube when visualized under UV light. The intensity of
fluorescence is directly proportional to the extent of oxygen depletion.
• MGIT tubes may be incubated at 37ºC and read manually under a UV
light or entered into a MGIT 960 instrument where they are incubated
and monitored for increasing fluorescence every 60 minutes.
• In case of M. tuberculosis, at the time of positivity, there are
approximately 105 – 106 colony forming units (CFU) per ml of medium.
• The instrument declares a tube negative if it remains negative for six
weeks (42 days).

Drug susceptibility Testing
• Two MGIT tubes are inoculated with the test culture. A known
concentration of a test drug is added to one of the MGIT tubes, and
growth is compared with the MGIT tube without the drug (growth
control). If the test drug is active against the isolated mycobacteria,
it will inhibit the growth and thus there will be suppression of
fluorescence, while the growth control will grow uninhibited and
will have increasing fluorescence. Growth is monitored by the
BACTEC 960 instrument which automatically interprets results as
susceptible or resistant.
• The BACTEC MGIT 960 susceptibility testing for Streptomycin (S),
Isoniazid (I), Rifampin (R) and Ethambutol (E) is called SIRE.
• Once the test is complete (within 4 to 21 days), the instrument will
indicate that the results are ready. The instrument printout
indicates susceptibility results for each drug.

• Pyrazinamide (PZA) Susceptibility Testing
Susceptibility testing against PZA is always carried
out at a lower pH of the medium, since pyrazinamide
(PZA) is active only at the low pH in vitro.
• The MGIT 960 medium is a modified 7H9 broth with
a reduced pH of 5.9. The detection of growth is
achieved by the oxygen sensor at the bottom of the
tube in the same way as the one in the regular MGIT
tube, and the principle of the detection of resistance
is the same as for SIRE drug susceptibility testing.

Liquid Culture and DST
• The BACTEC MGIT 960 susceptibility testing is available for
following drugs with the following critical concentration of drugs
recommended in the medium.
• Streptomycin -------------- 1.0 μg/ml of medium
• Isoniazid -------------------- 0.1 μg/ml
• Rifampin -------------------- 1.0 μg/ml
• Ethambutol ----------------- 5.0 μg/ml
• PZA --------------------------- 100 μg/ml
• Amikacin -------------------- 1.0 μg/ml
• Capreomycin --------------- 2.5 μg/ml
• Kanamycin ------------------ 2.5 μg/ml
• Ofloxacin -------------------- 2.0 μg/ml
• Moxifloxacin ----------------- 1.0 μg/ml
• Ethionamide --------------- 5.0 μg/ml
• Para-amino salicylic acid- 4.0 μg/ml
• Clofazimine ------------------ 0.5 μg/ml

 Other systems based culture
1. Microscopic observation drug susceptibility(MODS) -an inverted light
microscope
2. Nitrate reductase assay
3. Colorimetric Redox indicator methods - under evaluation
4. Thin layer agar
• Applicable to clinical sputum samples - performance comparable to
genetic methods, in their sensitivity and specificity
• The mean time to results 21 to 23 days

MODS in detection of drug resistance
• The microscopic-observation drug-susceptibility
(MODS) assay is a low-cost alternate to the
detection of drug resistance. By using Middle
brook 7H9 broth culture containing
antituberculous drugs, sputum is directly
inoculated, and growth (seen as cord formation) is
detected using an inverted light microscope. In
Ethiopia, MODS detection of MDRTB was excellent
with sensitivity and specificity of 95 and 100%,
respectively, when compared with the MGIT 960
system . The time to detection has been shown to
be 7 days and similar to the MGIT 960Dr.T.V.Rao MD 17

Colorimetric redox indicator methods
• Principal of CRI methods is the reduction of a
coloured indicator which is added to the culture
medium after cultured M. tuberculosis have been
exposed to the test antibiotic .
• Drug resistance is detected by a change in colour
of the indicator, which is directly proportional to
the number of viable mycobacteria remaining in
the medium after exposure to the antibiotic.
• Different indicators are
-tetrazolium salts XTT and MTT
-redox indicators Alamar blue and resazurin .

Nitrate Reductase Assay (NRA)
• Based on MTB’s ability to
reduce nitrate to nitrite
• Simple
• Sensitive detection of small
amount of metabolic
biproductimproves
turnaround time

Thin Layer Agar (TLA)
• Direct inoculation of
patient specimens –
detection & DST
• Solid media –easier to
manipulate
• Microcolonydetection –
faster turnaround time
• Average positive results
–11 days

Non commercial Liquid Culture and DST
• Although these methods are promising as
they allow the use of inexpensive materials
and give turnaround times similar to liquid
culture, these tests are not well standardized,
and require extensive training and
optimization before routine clinical use.

Nucleic acid amplification tests
• The majority of molecular tests have been focused on
detection of nucleic acids, both DNA and RNA, that are
specific to Mycobacterium tuberculosis, by
amplification techniques such as polymerase chain
reaction (PCR); and detection of mutations in the
genes that are associated with resistance to anti
tuberculosis drugs by sequencing or nucleic acid
hybridization. Recent developments in direct and rapid
detection of mycobacteria, with emphasis on M.
tuberculosis species identification by 16S rRNA gene
sequence analysis or oligohybridization and strain
typing, as well as detection of drug susceptibility
patterns, all contribute to these advances.

Nucleic acid amplification
techniques- NAAT
• amplify target nucleic acid regions - uniquely the M. tuberculosis complex. 16S rRNA gene
sequence analysis
• Directly used on clinical specimens
• Categorized as Commercial kits
In-house assays
• Commercial kits include
The Amplicor MTB tests
The Amplified MTD test and
The BD ProbeTec ET assay
• In-house tests are
Laboratory-developed Polymerase chain reaction (PCR) assays

Gene expert
 The Xpert MTB/RIF system is a recently developed TB-
specific application, designed for the GeneXpert platform,
to detect M. tuberculosis as well as rifampicin resistance-
conferring mutations directly from sample, in an assay
providing results within two hours.
 GeneXpert, the test device platform, was launched by
Cepheid in 2004.
 In December 2010, the World Health Organization (WHO)
endorsed Xpert for the rapid and accurate detection of TB,
particularly among HIV and people suspected of having
MDR-TB

Gene Xpert models
There are different gene
xpert instruments
1. The gene xpert 1
instrument consist of 1
module (1 site) to
process 1 sample.
2. The gene xpert 4
instrument consists up
to 4 modules for
processing up to 4
samples.
3. The gene xpert 16
instrument has up to 16
modules.
4. The gene xpert 48
instrument has up to 48
modules.

• The sample are prepared and
processed in a single use assay
specific cartridges.

Limitations
1. Careful compliance to the instructions in this insert is necessary to
avoid erroneous results.
2. A positive test result does not necessarily indicate the presence of
viable organisms. It is however, presumptive for the presence of
MTB and Rifampicin resistance.
3. Test results might be affected by antecedent or concurrent
antibiotic therapy. Therefore, therapeutic success or failure cannot
be assessed using this test because DNA might persist following
antimicrobial therapy.
4. Mutations or polymorphisms in primer or probe binding regions
may affect detection of new or unknown MDR-MTB or rifampicin
resistant strains resulting in a false negative result.
5. Cost -The cost of the device is Rs 10 lakhs with annual maintenance
(Rs 1 lakh) and with each test cartridge costing Rs 1000.
6. Continous supply of electricity is needed for operation of
instrument.

Line probe assay
• Molecular Genetic Test Systems for the
Detection of Mycobacterium Tuberculosis
Complex and its Resistance to First- and
Second-Line Anti-TB Drugs from
• Decontaminated Smear-Positive Pulmonary
Specimens and Culture Samples:
• GenoType MTBDRplus
• GenoType MTBDRsl

• GenoType MTBDRplus is based on the
DNA•STRIP technology and permits the
simultaneous molecular genetic identification of
• The M. tuberculosis complex
• Its resistance to rifampicin by the detection of
the most common mutations in the rpoB gene
• Its resistance to isoniazid by the detection of the
most common mutations in the katG gene and
inhA gene
from smear-positive pulmonary clinical
specimens or cultivated samples.

• GenoType MTBDRsl is based on the DNA•STRIP technology and
permits the simultaneous molecular genetic identification of
• the M. tuberculosis complex
• its resistance to fluoroquinolones like ofloxacin and moxifloxacin by
the detection of the most common mutations in the gyrA gene
• its resistance to the injectable antibiotics ( kanamycin, amikacin
and capreomycin) by detection of the most common mutations in
the rrs gene
• its resistance to the first-line drug ethambutol by detection of the
most common mutations in the embB gene from smear-positive
pulmonary clinical specimens or cultivated samples

• Mutations detectable by Mutiple Drug Resistance (MDR)
assay for first line drugs
Drug gene total
Rifampicin rpoB 7
mutations D516V,D526Y,D526D,D531L,H526Y,H526D,S531L
Isoniazid(INH) inhA 3
c-15t, t8c, t8a
katG 6
S315T, T308P,Q295D,S315N,T324P,D329P

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