Introduction
·
Mycobacterium tuberculosis (MTB) is the
most infectious
·
Infection Estimated 10.0 million people
in 2019 globally,
·
leading to 1.4 million deaths in 2019
·
Diagnosis of tuberculosis is a
challenging task, especially in-Paucibacillary conditions, HIV patients and
Pediatric population.
Field of TB diagnostics
has seen advances in the form of new molecular tests.
As nucleic acid
amplification tests (NAATs)
·
Low complexity NAATs -
Gene Xpert MTB/Rif Gene Xpert MTB/ Rif ultra,
TrueNat, Xpert XDR etc.
·
Moderate complexity NAATs -
Genotype MTBDplus, BD Max MDR, Abbott Real Time MTB
etc.
·
High Complexity Sequencing -
PSQ (Pyrosequncing), TNGS ( Targeted Next Generation
Sequencing), WGS (Whole Genome sequencing)
Recent Advances in Microscopy
According to WHO
LED microscopy is more sensitive than conventional
fluorescence and light microscopy.
According to a study by Shenai et al,
LED microscopy
Sensitivity of 78.3%
Specificity of 92.0%
Mean time per smear examination- 1.41
minute
ZN stain - 2.48
minutes
Advances in culture
BACTEC MGIT 960 Culture System
Within 10 days-
Recovery of Mycobacterium,
Drug susceptibility testing - In shorter time span.
While LJ culture as – 8 weeks
Culture media-
Middlebrook 7H9 broth
But major limitation
-
High cost of equipment
-
Availability only at limited tertiary
care centers.
MODS (Microscopic-Observation
Drug-Susceptibility) Assay
based on three
principles-
1.
MTB grows faster in liquid medium,
2.
Cord formation can be visualized
microscopically
3.
Direct drug-susceptibility testing with
bacterial growth.
4.
Detect MDR TB
In a meta-analysis of
12 studies, Minion et al have shown
MODS (Microscopic-Observation Drug-Susceptibility)
-sensitivity of 92%
-specificity of 96%
The average
contamination rate -6.6%
TAT
- 9.2 days Thus,
~MODS is an
inexpensive,
~Rapid alternative to
conventional method for DST
ANTIGEN DETECTION
METHODS
Targeted antigen is
Lipo-arabinomannan (LAM)
For pulmonary TB,
Sensitivity - 2 to 100%
Specificity - 33 to 100
ž Also
called LAM urine assay.
ž LAM
is a major constituent of the MTB cell wall.
ž Its
presence in urine indicates activation of MTB and can be tested by ELISA.
ž It
uses as a rapid point of-care test ,
ž Research
to improve their performance is urgently needed.
MOLECULAR ADVANCES
Molecular TB DST Tests
by complexity.
Genes responsible for Drug Resistance
|
Drug |
Gene Target |
|
Rifampicin |
rpoB |
|
Isoniazid |
InhA, katG |
|
Fluoroquinolones |
gyrA, gyrB |
|
SLID (KAN, AMK, CAP) |
Eis, rrs |
1. Gene
Xpert MTB/RIF
·
Developed – in 2010
·
fully automated
·
Detects both – MTB & Rif R in 2 hrs
·
Extracts, purifies, Concentrates,
amplifies & target specific sequence of the rpoB gene,
·
RpoB
gene responsible for- RIF resistance
·
LOD- 130-150cfu/ml
·
Minimal biohazard and very little
technical training
·
sensitivity of 99.1%, specificity of
100%.
2. Gene
xpert mtb/rif ultra
·
Similarly Detects MTB and RIF resistance
·
Overcome the limitations of Old Xpert
MTB/RIF
·
Detects silent mutations improving the
detection of RIF resistance.
Higher
sensitivity (97.92%)
lower
specificity (98%)
3. TrueNat
·
Chip-based nucleic
acid amplification test in the detection of MTB (Developed by the India firm
MolBio Diagnostic Pvt Ltd Goa
·
Steps followed-
·
DNA
extraction using Trueprep - MAG protocol.
· Real-time PCR on chip/Real-time PCR on ABI 7500
TrueNat VS CBNAAT
CBNAAT detected 7 negative specimens
whereas TrueNat detected 13 negative Specimens
|
TrueNat |
CBNAAT |
|
Sensitivity- 94.05 |
Sensitivity- 86 |
|
Specificity-42.86 |
Specificity- 99 |
|
NPV-97.53 |
NPV- 96 |
|
PPV-23.08 |
PPV- 98 |
|
Cartridge based LOD- 100 cfu/ml |
Chip based LOD- 130-150 cfu/ml |
4. Xpert
XDR
·
Another PCR-based cartridge
designed to run on the GeneXpert
·
Detection of mutations associated
with resistance to multiple first and second-line TB drugs extensively
drug-resistant TB (XDR-TB).
5. Probe
Based with specific mutation LPA
First Line probe assay- 2008
·
e.g. Genotype MTBR
plus
·
Principle- PCR, Hybridization
·
Use- Diagnosis of RIF and INH resistance
·
Sensitivity- 98%
·
Specificity- 99%
·
Target setting
of use- Reference laboratory
·
TAT- 5 hrs
Second Line probe assay
·
Developed in 2016
·
Principle- PCR
hybridization
·
Use- Diagnosis of
FLQ and SLID resistance
·
Sensitivity- 86%
·
Specificity- 99 %
·
Target setting
of use- Reference laboratory
· TAT- 5 hrs
Limitations
of LPA
·
Imperfect sensitivity for pauci
bacillary disease
·
Open assay increase contamination
potential
·
Requires manual interpretation
·
Low throughput
·
Inability to indicate all the SNPs
responsible for resistance.
6. Pyrosequencing
(PSQ)
Novel real time DNA sequencing
- Detects XDR TB
- Detects MTB resistance to INH, RIF, and FQ and
SLIs (KAN and CAP)
7. Whole genome sequencing
·
Full genome sequencing
·
No prespecified targets needed
·
Detects rare mutations and
heteroresistance
·
Reliable and low-cost DNA
extraction method
·
1 mL of early positive MGIT cultures
that is enough for the WGS sequencing & predict antibiotic resistance in
clinical samples
Disadvantages of WGS
·
Requires culture isolates
·
Slower than targeted NGS
·
Complicated bioinformatics
·
Expensive
8. Targeted NGS
·
Sequence directly from sample
·
Large number of gene targets
·
Less expensive than WGS
·
Simpler bioinformatics and storage
·
Detects rare mutations and
heteroresistance
Targted sequencing work flow
Whole genome sequencing vs Targeted NGS
|
WGS |
NGS |
|
Entire genome sequenced |
Large no of specified targets
adaptable to new genes, scalable |
|
Needs culture isolates |
Directly on sputum |
|
Comprehensive solution |
Less information than WGS |
|
Complicated Bioinformatics |
Simpler Bioinformatics on cloud |
|
Detects rare variants |
Knowledge OF targets needed |
|
Slower TAT- 2-3 week |
Faster TAT – 2-3 days |
UREASE BREATH TEST
·
For rapid diagnosis of TB Metabolic
pathway detection
·
Rapid and effective new tools for
TB that can improve TB diagnostics for children and HIV-infected patients.
·
Metabolic breath tests have
advantages because these are safe and rapid tool for drug efficacy evaluation
during clinical trials.
·
Trials are currently ongoing.
THANK YOU…..