About newer diagnostics of tuberculosis

1. NEWER DIAGNOSTIC METHODS
FOR TUBERCULOSIS
Presenter Dr. Shweta Anand
Moderator Dr. V. P. Myneedu
NITRD

2. INTRODUCTION
TUBERCULOSIS:
TB is an infectious disease caused mainly by the bacillus
Mycobacterium tuberculosis, a small, aerobic, nonmotile gram
positive bacillus.
MODE OF SPREAD:
Infectious aerosol droplets 0.5 to 5.0 µm in diameter.
A single sneeze can release up to 40,000 droplets. Each one
of these may transmit the disease, since the infectious dose is
very small (the inhalation of fewer than 10 bacteria may
cause an infection).

3. GLOBAL BURDEN OF DISEASE
There were an estimated 10.4 million new cases of TB in 2015; 56%
males, 34% women, 10% children
An estimated 1.2 million of the 10.4 million people were HIV-positive.
1.79 million died from the disease (including 390 000 deaths among
HIV positive people)
 An estimated 4,80,000 people developed MDR- TB, RR-TB: 1,00,000;
new 3.9%; previously treated 21%
 Case fatality ratio is 17%
Global TB report 2016

4. MORTALITYINCIDENCE

5. BURDEN OF DISEASE IN INDIA:
 about 40% of the Indian population is infected with TB
bacteria, the vast majority have latent rather than active TB.
 Incidence of 217 new cases per lakh and 8.6 cases per lakh
among HIV TB
Tuberculosis Mortality of 36 per lakh.
 Estimated MDR cases are 79,000;
New- 2.5% (2.1–3.1)
Previously treated- 16% (14–18)
Global TB report
2016

6. INCIDENCE MORTALITY

8. END TB STRATEGY

9. NEED FOR NEWER
DIAGNOSTICS FOR
TUBERCULOSIS
Many people with TB do not have access to
adequate initial diagnosis.
In 2013, 58% of the 4.9 million pulmonary TB
patients notified globally were bacteriologically
confirmed via a WHO recommended test, including
rapid tests (Xpert MTBRIF).
Rest were likely managed on the basis of clinical
suspicion or non specific tests.
Access to diagnosis is particularly challenging in
people with MDR TB and in children with TB.

10. Sputum collection and Microscopy
Culture-based Diagnostics
Molecular detection
Cellular response- to detect latent and
latent to active progression.
Breath biomarker – detection
Antigen antibody and biomarker detection
Radiology
Outline: TB Diagnostics Methods

12. SPECIMEN COLLECTION AND
MANIPULATION
A persistent problem is that the patient cannot provide
sufficient volume of sputum specimen for sputum testing.
A suboptimal specimen would be:
- Limited volume
- Compromised sample(excessive
saliva)
Presently patient gives induced sputum via saline spray.
PLHIV also produces pauci-bacillary specimens.

13. Products which improve specimen collection:
- Lung Flute
- OMNI gene SPUTUM
- PS(MTM)
- Deton Corp
LUNG FLUTE
• It is non- invasive
• It is a small plastic device held by the user to the lips and
exhales into it.
• This creates vibrations in the lungs that help to loosen and
liquify sputum in the alveolar cavities. Hence, person is able
to produce more sputum.
• US-FDA approved CE-IVD marked.
• Sensitivity 84.3% as compared to hypertonic saline(78.4%)
SPUTUM COLLECTION CONTD…

14. SPUTUM COLLECTION CONTD…
DETON CORP
• This product is in the mid- development stage.
• Patient coughs into a 1L bag and the micro- droplets remain
suspended in the airspace of the collection bag due to their
very small size.
• Collected air is then forced through a device engineered to
impact the particles onto a collection surface and used for
diagnostic tests.

15. OMNI gene SPUTUM
• Processes the sputum
• Liquifies and de- contaminates the sample
• Allows it to be transported without cold chain(upto 8 days)
A study in Nepal(GENETUB) demonstrated significant reduction in culture
contamination and improvement in average time to positive culture result.
PS(MTM)
• It contains chemical de-naturants that permits the lysis of MTB
cells with subsequent stabilization of MTB DNA without cold chain.
• Additional benefit is that not all of the genetic material is used for
a single test. There is extra material for secondary testing also.
SPUTUM COLLECTION CONTD…

16. SMEAR MICROSCOPY
METHODS: (commonly used)
 Zeihl neelsen technique (hot acid fast stain)
 Kinyoun technique (cold acid fast stain)
 Fluorescent technique
 Vital staining technique (differentiate between live and
dead bacilli)

18. Acid Fast Bacilli under
Fluorescent Microscope
Dr.T.V.Rao MD 18

20. Advances in microscopy
LED Fluorescent Microscopy:
• Fluorescence + 10% sensitivity vs ZN
≥ 46% less time
• Equipment less expensive, more durable, less
maintenance
• No hazardous components, bulb life 10+ yrs, no
warm-up time
• Does not require a dark room
Disadvatages: Lack of sensitivity.
Tomans Tuberculosis 2nd edition

21. AUTOMATED MICROSCOPY
TBDX SYSTEM (Signature mapping medical sciences)
• Utilizes novel software algorithms to scan high resolution digital
images of fluorescent smears to automatically score the fluorescent
bodies.
• The system integrates a high-quality fluorescent microscope and the
software analyses digital images of each field.
• takes 5 mins to analyze one slide.
• TBDx can be adjusted for highly positive slides in order to expedite
testing.
• Overall senistivity 79.8%, specificity 78.9%
A study in South Africa comapared performance of TBDx to 40 yr
veterans of smear microscopy using culture as reference standard, TBDx
98% sensitivity on SSM+/C+; 40% on SSM-/C+

22. TBDx system showing the automated slide loader (left),
FM with digital camera and
automated stage (centre), and laptop to operate the
reader and employ the scoring
algorithm (right)

23. AUTOMATED SMEAR MICROSCOPY
READER
• Currently under development
• Becton Dickinson is manufacturing
• Fully automated
• Also incorporates the staining procedure for detection of AFB
in sputum.
• Esp useful for microscopy centers or lower tier facilities.
• A slide-reading algorithm scores the reading of slides,
eliminating the subjectivity associated with manual
examination.
• relatively unskilled workers.
• takes about five minutes hands-on time
• sensitivity of the prototype is similar to that of LED-FM
performed by a skilled microscopist.

24. Automated smear microscopy reader
in development by Becton Dickinson

25. Mycobacterial culture method
• Gold standard for diagnosis
• Can detect as few as 10 viable bacilli per millimeter of sample.
(5000-10000 SSM, 100 NAAT)
• Distinguishes even between individual members of MTBC by
biochemical and phenotypic testing.
• Permits DST.
• Can take as long as 8 weeks.
• Requires level 3 biosafety measures.
• Sensitivity 80- 85%
Specificity 98%.

27. Lowenstein –Jensen medium
Egg based media with addition of salts, 5 % glycerol,
Malachite green
Advantages:
- Specificity about 99 %
- More sensitive
- Can differentiate between TB complex &
NTM using biochemical reactions
- Sensitivity tests for anti-tuberculous drugs
( St, INH, Rif., E)
Disadvantages:
-Slowly growing ( up to 8 weeks )
27
Sharma Mohan 2nd edition

29. ▪ Sauton’s Media

30. • semi-automated system
• Medium –modified middle brook 7H9 medium
• Palmitic acid is radiolabeled with C14
• Contamination is controlled by addition of
P- Polymyxin B
A- Amphotericin b
N- Nalidixic acid
T- Trimethoprim
A- Azlocillin
• Reconstituted with polyoxyethylene solution
• If viable mycobacteria present in vial radioactive palmitic acid is
metabolized and radioactive CO2 is liberated in to gaseous phase
which is measured with β counter.
BACTEC TB-460

31. (MGIT) Mycobacterial Growth Indicator
Tube
• Rapid Method.
• Consists of round bottom tubes
containing 4 ml of modified
Middlebrooks 7H9 broth which has
an oxygen sensitive fluorescent
sensor at the bottom.
• When mycobacteria grow, they
deplete the dissolved oxygen in the
broth & allow the indicator to
fluoresce brightly in a 365nm UV
light.

32. BacT/Alert 3D(MB/BacT)
• Modified Middlebrook 7H9 medium is used
• A mixture of OADC enrichment and polymyxin B,
amphotericin B, nalidixic acid, trimethoprim,
vancomycin and azlocillin
• Decontamination step needed
• Incubator and reader is combined in a single system
• Has CO2 sensor at the bottom, CO2 sensor is impacted
by light, reflected rays are monitored by photodiode

33. MB/BacT

35. versaTREK(ESP system-2)
• Modified middlebrook 7H9 medium
• OADC enrichment
• Two kind of anti-mycobacterial mixture
AS PVNA
Polymyxin B, azlocillin,
fosfomycin, nalidixic acid,
amphotericin B
Polymyxin B, vancomycin,
nalidixic acid, amphotericin B
Used when sample is sterile with
low risk of contamination
Heavily contaminated sample
with high risk of contamination

36. versaTREK(ESP system-2)contd…
Based on the detection of pressure
changes within the headspace above
the broth culture medium in a sealed
bottle i.e. either gas producion or gas
consumption by the microbes.

37.  Microscopic Observed Drug Susceptibility
(MODS)
 Thin Layer Agar (TLA)
 Nitrate Reductase Assay (NRA)
 Colorimetric redox indicator (CRI)
 Mycobacteriophage-based assays
Novel Culture-based Diagnostics

38. MODS
A microcolony method, based on direct inoculation of
patient specimens to drug-free and drug-containing liquid
media followed by microscopic examination of early culture
growth.
Growth of M. tuberculosis is identified by typical cord
formation under an inverted light microscope.
Growth in drug-free media indicates a positive culture;
growth in both drug-free and drug-containing media
indicates resistance.

39. MODS
• MODS is highly sensitive (98.0%) and specific (99.4%) for
the detection of rifampicin resistance.
• Less sensitive for isoniazid (sensitivity 91.4%, specificity
97.7%).
• MODS requires additional staff skills
• MODS is as expensive as CRI, less expensive than
commercial liquid culture, and more expensive than TLA or
the NRA.
• Average positive results ~9 days

41. • Microcolony direct method on solid culture media
using a standard light microscope to
simultaneously detect M. tuberculosis complex and
indicate isoniazid and rifampicin resistance.
• Growth on drug-free media indicates a positive
culture; growth on both drug-free and drug-
containing media indicates resistance.
• Average positive results –11 days
• Sensitivity 100%; Specificity 100%
Thin Layer Agar (TLA)

42. Thin Layer Agar (TLA)
TLA appears to be a promising
diagnostic tool for rapid DST and
further research is
encouraged.

43. • Solid culture technique
• Based on the capacity of M. tuberculosis to reduce nitrate to
nitrite.
• Detected by adding a specific reagent (Griess reagent) to
conventional Löwenstein-Jensen (LJ) medium into which 1 mg/m
of potassium nitrate (KNO 3 ) has been incorporated.
• The reduction of nitrate is detected by a coloured reaction.
• Rifampicin resistance sensitivity 97%
specificity 100%.
• Isoniazid resistance sensitivity 97%
specificity 99%.
Nitrate Reductase Assay (NRA)

44. Colorimetric redox indicator (CRI)
• Indirect methods based on the reduction of a colored indicator added to
liquid culture medium in a microtiter plate after M. tuberculosis has been
exposed in vitro to different antibiotics and different drug concentrations.
• Resistance is detected by a change in color of the indicator, which is
proportional to the number of viable mycobacteria in the medium.
• growth indicators used are:
• tetrazolium salts
• redox-indicators Alamar blue and resazurin.
• Rifampicin sensitivity 98.0%
specificity 99.0%
• Isoniazid sensitivity 97.0%
specificity 98.0%

45. DST Rapid colorimetric

46. PCR
LAMP
TMA / NAA
Ligase chain reaction
GENOTYPIC METHODS

47. Polymerase Chain Reaction (PCR)
• Earlier 65 Kd antigen (HSPs) Heat shock protein was used as a
target protein
•This gene is identical in all species of mycobacteria.
•Therefore unsuitable for detecting M.tb, particularly in areas where
species like M. avium or M.kansasii are prevalent.
•IS6110: is a transposon which are self replicating stretches of DNA.
•This sequence has been 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

48. Polymerase Chain Reaction (PCR)
•Role in pulmonary TB :
•Detects nearly all smear +ve and culture +ve cases.
•Useful technology for rapid diagnosis of smear –ve cases of
active TB.
•Able to identify 50-60% of smear -ve cases; this would reduce
the need for more invasive approaches to smear −ve cases.
Distinguishes M.tb from NTM in smear +ve cases as IS6110
sequence is not found in NTM.
•Should not be used to replace sputum microscopy.
•Sensitivity, specificity, & PPV for PCR is 83.5%, 99% & 94.2%
respectively.

49. Available semi automated NAATs
• Eiken loopamp MTBC assay (Japan)
LAMP
Better sensitivity as compared to SSM
• Genedrive MTBC assay (UK)
Currently, the test assays only for diagnosis of MTBC with a control
reaction, but a third test to genotype MDR TB via RIF resistance is in the final
phases of internal validation by the company
a genotyping assay targeting rpoB via the RRDR.
Sensitivity and specificity >95%
• Truelab RealTime micro PCRSystem (India)
a sensitivity of 99.6% with SSM+/C+ samples and 75.6% with
SSM-/C+
Testing time 1hour
• EasyNAT TB assay (china)
• analytical sensitivity of 10 cfu/mL and 100% specificity
• clinical sensitivity for SSM+/C+ of 98.1% and SSM-/C+ of 77.8% with a specificity of 89.2% versus

50. LAMP
•Loop-mediated isothermal amplification.
•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 liquid medium or solid
medium.
•This method employs a DNA polymerase and a set of four
specially designed primers that recognize a total of six distinct
sequences on the target DNA.
• Species-specific primers were designed by targeting the gyrB
gene.
• Simple procedure, starting with the mixing of all reagents in a
single tube, followed by an isothermal reaction during which the
reaction mixture is held at 63°C.
• 60-min incubation time

51. Ligase Chain Reaction
• It is a variant of PCR, in which a pair of oligonucleotides are
made to bind to one of the DNA target strands, so that they
are adjacent to each other.
• A second pair of oligonucleotides is designed to hybridize to
the same regions on the complementary DNA.
• The action of DNA polymerase and ligase in the presence of
nucleotides results in the gap between adjacent primers
being filled with appropriate nucleotides and ligation of
primers.
• It is mainly being used for respiratory samples, and has a
high overall specificity and sensitivity for smear +ve and –ve
specimens.

52. Line probe assay
•Line probe assay uses the reverse hybridization technology
with multiple differently specific DNA probes.
VERSION 1:
Two commercial assays available
•Genotype MTB DRplus
•INNO-LipA Rif.TB
•Validated for use directly from smear-positive
sputum (MTBDRplus) or from TB cultures
•Manual and automated system
•rpoB for rifampicin resistance
•katG and inhA for isoniazid resistance (MTBDRplus)``

53. MTBDRsl v2.0.
• Increased sensitivity via an improved PCR
• May be used on SSM-/C+ sputum
• Effective in second line drug resistance
• Test to rule in drug resistance but not to rule out drug
resistance
• Has a total of 27 probes for the detection of resistance
to second line drugs
• New target genetic regions are: eis (KAN) gyrB (FLQ)

56. Xpert MTB/RIF

57. Xpert MTB/RIF Contd…
• TB-specific automated, cartridge-based nucleic amplification
assay
• currently unique in its simplification of molecular testing - has
fully integrated and automated sample preparation,
amplification and detection required for real-time polymerase
chain reaction
• Xpert MTB/RIF detects M. tuberculosis as well as rifampicin
resistance-conferring mutations directly from sputum
• results within 100 minutes.

58. Xpert MTB/RIF Contd…
• The molecular beacons which target the rpoB gene cover all the
mutations found in >99.5% of all rifampicin resistant strains
• no cross-reactivity with non-tuberculous mycobacteria
• The sample reagent added in a 2:1 ratio to sputum was shown
to kill >6 log10 cfu/ml of M. tuberculosis with 15 minutes of
exposure, and to render >97% of smear-positive samples
negative by LJ culture.
• So no need for biosafety cabinet

59. Xpert MTB/RIF Contd…

60. Xpert MTB/RIF Contd…
• 92.2% of culture-positive patients are detected by a single
direct Xpert MTB/RIF test.
• Sensitivity of a single Xpert MTB/RIF test in smear-
negative/culture-positive patients was 72.5% and increased to
90.2% when three samples were tested.
• Xpert MTB/RIF specificity was 99%.
• Xpert MTB/RIF detected rifampicin resistance with 99.1%
sensitivity and excluded resistance with 100% specificity.

61. WHO Recommendations
• Xpert MTB/RIF should be used as the initial diagnostic test in
individuals suspected of MDR-TB or HIV-associated TB (strong
recommendation)
• Xpert MTB/RIF may be used as a follow-on test to microscopy in
settings where MDR and/or HIV is of lesser concern, especially in
smear-negative specimens (conditional recommendation,
recognising major resource implications).
• Xpert MTB/RIF technology does not eliminate the need for
conventional microscopy culture and DST
• Xpert MTB/RIF is suitable for use at district and sub-district level,
outside of conventional laboratory settings

62. Latest development
Cepheid Inc. is currently developing two new MTB assays to be hosted
on the Xpert® platform:
• the Xpert® MTB/RIF Ultra
• XDR assays.
The Xpert® MTB/RIF Ultra:
• The Ultra is designed to have an LOD similar to that of culture to
more accurately diagnose PTB from paucibacillary specimens.
• a new assay targeting MTBC specific regions, the insertion
sequences IS6110 and IS1081 has been developed
• A process control using B. globii is still included to ensure
appropriate test performance.
• An assay to target rpoB is also incorporated

63. The Xpert® MTB/RIF Ultra contd…
• the Ultra cartridge uses double the volume of input sample
material as compared to the MTB/RIF cartridge, with 50 µL
rather than 25 µL.
• The LOD for MTBC using this format is claimed to be only 5
cfu when testing analytical sensitivity. The current Xpert®
MTB/RIF assay has a sensitivity of ~150 cfu/mL sample.
• clinical sensitivity for SSM-/C+ specimens is >90% with
100% specificity.

64. XDR ASSAY
• The XDR assay is designed to be a reflexive test when an
Xpert® (or Ultra) MTB/RIF positive test indicates an infection
with RIF-resistant MTBC.
• The XDR assay will further genotype common resistance
alleles to INH, FLQ and AMG.
• no performance data available for the XDR assay.
Modifications to Xpert® instrument to operate these new
assay cartridges will need only software upgrades and the
recalibration of the optics system. New machines are not
required and thus these new assays can be introduced for
use on the Xpert® platforms already implemented.

65. Current and emerging automated, semi-modular or non-
integrated TB NAATs; their intended laboratory location and
release date

66. NAAT IN DEVELOPMENT
Alere™ q instrument for TB
testing, currently in
development
Cepheid Inc. GeneXpert® Omni: a
standalone tool, currently in
development, for the
independent processing of the
Xpert® test cartridges at POC

67. NAAT IN DEVELOPMENT CONTD…
GenePOC
automated test
device
KGI TBDx system:
instrument (left)
and test cartridge
(right)

68. NAAT IN DEVELOPMENT CONTD…
Point of Need (PON)
technology in development by
Qiagen: a schematic rendering
of test cartridge design (left)
and an image of a prototype
instrument (right)
Rendering of the collection unit
of the Wave 80 Biosciences
EOSCAPE-TB System

69. BIOMARKERS TO DETECT ACTIVE TB
• It uses non invasive or minimally invasive specimens like breath,
urine, finger stick.
• Determine TB LAM Ag rapid assay:
It is an immuno chromatographic strip that targets the LAM Ag in
urine via an antibody capture and detection method on a nitro
cellulose strip.
This assay is not only specific for MTBc but also detects other NTMs.
It indicates all sites of TB because LAM is released in the blood and
ultimately expelled by urine.
Duration of test is 25 mins, minimal training required, product stable
for 15 months at 30C.

70. BIOMARKERS TO DETECT ACTIVE TB
CONTD…
Lawn et al., noted that the test performance improves with
increasing severity of illness and more advanced
immunosuppression.
• Sensitivity incrementally improved from 4% to 76% as the
CD4 count decreased from > 200 to < 50.

71. VOLATILE ORGANIC COMPOUNDS
• Assessing VOCs via simple breath tests for rapid screening of
TB infection.
• TB Breathalyzer:
-Rapid bio-sensor systems limited.
-Detects active TB bacilli in cough sample in less than
4 minutes.
-Patient coughs into a cough collector which is then placed
into a portable optical reader that interrogates the
cough sample via MTB specific Ag detection.
-Software outputs TB +ve or TB –ve result on LCD display.
-Specificity 79%

72. TB Breathalyser from Rapid Biosensor Systems Ltd:
cough collector (left and centre) and
sensor to detect the presence of MTBC-specific antigens
(right)

73. Antibody detection by ELISA
Diagnosis by antigen and antibody detection is not approved in
India.

75. DIAGNOSIS OF LATENT
TUBERCULOSIS
• Tuberculin skin test(TST)
• Interferon gamma release assay(IGRA)
• NO GOLD STANDARD FOR THE DIAGNOSIS OF
LATENT TUBERCULOSIS

76. IGRAs
commercial kits are available
1. QuantiFERON-TB GOLD (Cellestis Ltd, Australia)
2. T SPOT-TB (Oxford Immunodec, oxford, UK)
3. Immu-check TB Platinum (Immunoshop India Pvt Ltd)
4. TB-IGRA (Beijing Wantai Biological Pharmacy Enterprise
Co. Ltd China)
5. ASACIR TB (Haikou VTI Biological Institute (China)
Of these assays, only the QFT plus and T-SPOT® TB
assay have CE-IVD marking and US FDA approval

77. Tuberculin test

78. TST vs IGRAs
characteristics TST IGRAs
Estimated sensitivity 75-90% 80-95%
Estimated specificity 70-95% 95-100%
Cross reactivity with
BCG
yes Less likely
Cross reactivity with
NTM
yes Less likely
reliability Moderate high
Boosting
phenomenon
yes no
cost low high
Patients visit two one
result 2-3 days 1-2 days
Inter reader
variability
yes minimal

79. DIGITAL CHEST XRAY
• DCXR has potential use in both active and passive case finding
of TB and can integrate with other diagnostic tests such as Xpert
MTBRIF for rapid identification of active TB.
• Used as a screening tool.
• ADVANTAGES:
fast
low variable cost
sensitivity/ specificity = 90/80 (low HIV prevalence)
• DISADVANTAGES:
Insufficient trained staff to read the xray.
sensitivity/ specificity = 80/75 (medium and high HIV
prevalence)

80. COMPUTER AIDED DIAGNOSIS FOR
TB (CAD4TB)
• To overcome the disadvantage, the Delft imaging
systems(Netherlands) invested in the development of
software that automatically score each CXR image within
one minute.
• In Oct 2014, it released the 4th version of CAD4TB which
was also CE- certified in Q2 2015 for use with any DCXR
platform.
• CAD4TB now surpasses the performance of a trained
reader in the field or hospital and can be used for passive
and active case findings as well as prevelance surveys.
• It automatically analyzes the chest image and
abnormalities consistent with TB.
• It gives a score between 0 to 100 within one minute.

81. DIGITAL CHEST XRAY CONTINUED…
Advenio( Chandigarh India) is developing
automated imaging analysis software and has a TB
specific product riView TB.
This is similar to CAD4TB.
Advenio is also developing algorithms that will also
screen for pneumonia and silicosis to augment PTB
diagnosis.

82. DCXR (left) and CAD4TB interpretation
of lung abnormality (right)

83. Components from the EasyPortable DCXR that
highlight its portability for use in
non-hospital settings

85. THANK
YOU