MOLECULAR TESTS IN DIAGNOSIS OF TB

Dr. Sachin Sharma

2nd Year Resident
Dept. Of Respiratory Medicine
IPGMER & SSKM HOSPITAL
 TB remains a major public health issue
especially in developing nations due to the lack
of adequate diagnostic testing facilities.

 Diagnosis of TB has entered an era of

molecular detection that provides faster and
more cost-effective methods to diagnose and
confirm drug resistance in TB cases.
ROLE OF MOLECULAR
TESTS
 Crucial for earlier MTB and Drug Resistance
detection.

 Shortened time to treatment initiation.

 Improved retention in care.

 Improved treatment outcomes.

 The majority of molecular tests have been
aimed at the detection of MTB specific nucleic
acids, both in DNA and RNA, by doing Nucleic
Acid Amplification (NAA) using amplification
techniques such as Polymerase Chain Reaction
(PCR).

 Detection of genes mutation that are related

with the resistance to anti-TB drugs by
sequencing or nucleic acid hybridization.
 MOLECULAR ASSAYS

PROBE BASED ASSAYS SEQUENCE BASED

(NON-SEQUENCING) ASSAYS

Can detect wheather a gene Can provide information

Mutation is present but cannot regarding the nature of a
provide the sequence specific mutation and predict
information for the specific drug resistance with greater
mutation. accuracy.
 Result obtained from a probe-based assay
suggestive of resistance should be confirmed
with a sequence based assay or by culture.

 All molecular test for drug resistance must be

confirmed by culture.
 PROBE BASED ASSAYS
 Mainly based on NAA testing.
(Nucleic Acid Amplification)

 NAATs amplify a specific nucleic acid sequence

that can be detected via a nucleic acid probe
and the amplification technique mainly used is
Polymerase Chain Reaction (PCR).

 NAAT is used for rapid diagnosis

(usually within 24-48 hours).
PCR

ZZZZ
PCR
PROBE BASED ASSAYS
 Xpert MTB/RIF
 Xpert ULTRA

(CARTRIDGE-BASED NAAT)
GeneXpert Omni
 Xpert MTB/XDR
 TRUENAAT (CHIP-BASED)
 LINE PROBE ASSAYS (LPA)
 TB-LAMP
 CB-NAAT (GeneXpert)
 Works on Real-Time PCR.
 Simultaneously detects MTB
and resistance to RIFAMPICIN.
 Testing time- 2 hours.
 Requires uninterrupted power
supply and temperature control
setting.
 United States (Cepheid).
 Foundation for Innovative New
Diagnostics (FIND), supported
by the American National
Institutes of Health.
 Concentrates
Bacilli and remove
inhibitors
ACCURACY OF GeneXpert FOR VARIOUS
SAMPLES
SAMPLES SENSITIVITY SPECIFICITY

SPUTUM 88% (MTB Detection) 98%

95% (Rifampicin Resistance) 98%
68% (Smear Negative Cases)

CSF 81% 98%

LYMPH NODES 83% 94%

PLEURAL FLUID 46% 99%

GASTRIC LAVAGE 84% 98%

 ADVANTAGES DISADVANTAGES

Can detect MTB and RR at the same Requires stable uninterruptable

time. electricity.

Rapid (results in <2 hours) Operating temperature should not

exceed 30º C and cartridge must be
stored at <28º C.

High sensitivity and specificity when Cartridge’s shelf life must be monitored
compared to liquid culture. or are prone to being wasted.

Low biosafety level (BSL) requirements. Amplification from dead bacilli-Can not
be used to monitor TB treatment.

Minimal training of personnel. Instruments accuracy need to be

checked every year.

Can detect both PTB & EPTB. Blood or blood tinged sample cannot be
used.
Xpert MTB/RIF
 Endorsed by WHO: 2011
 Approved by FDA: 2017
 It only concentrate on single target for
amplification of TB detection: rpoB Gene.

 Resistance detection is by-Real Time PCR

where 5 probes binds to rpoB Gene.
 PCR Reaction: 25µL.

 Turn around time(TAT): 112 min.

 Load of detection(LOD): 131 cfu/ml.
Xpert ULTRA
 In 2017, WHO recommended Xpert ULTRA
the next generation of Xpert MTB/RIF for
diagnostic use.
 Uses the same GeneXpert platform.
 Developed to overcome the limitations of
Xpert MTB/RIF-
 Imperfect Sensitivity (particularly in smear
negative & HIV asso. TB).
 Better determination of Rifampicin Resistance.
 Divide the TB Bacilli load into-

 High
 Medium
 Low
 Very low
 Trace (New category-corresponds
to lowest bacillary burden)

 It also detects non-replicating or non viable

bacilli present particularly in patients with
recent history of TB, reducing the overall
specificity of ULTRA in high-burden TB
setting.
 It concentrate on multiple target for
amplification of TB detection-
 rpoB Gene
 Insertion Sequence: IS6110, IS1081.

 Resistance detection is by-Melting Curve

Analysis where 4 probes binds to rpoB Gene.
 PCR Reaction: 50µL.

 Turn around time(TAT): 65-87 min.

 Load of detection(LOD): 16 cfu/ml.
 Xpert MTB/RIF Vs Xpert ULTRA
Xpert MTB/RIF Xpert ULTRA

Amplification for TB Single target: Multiple target:

Detection rpoB core region rpoB core region
IS6110
IS1081

Resistance Detection Real Time PCR Melting curve analysis

(5 probes bind to rpoB (4 probes bind to rpoB
gene) gene)

PCR Reaction 25µL 50µL

Turn Around Time(TAT) 112 min. 65-87 min.

Load of Detection(LOD) 131 cfu/ml 16 cfu/ml

GeneXpert OMNI
 Small, portable, integrated,
battery operated device.
 Can use both Xpert MTB/RIF
and XpertULTRA Cartridges.
 Expands testing to
disseminated locations.
 Processes one sample at a
time, not suitable for high
attendence clinics.
Xpert MTB/XDR
 Works on GeneXpert platform.

 For- Isonazid, FQs, Ethionamide and SLI

resistance detection.

 Compared to phenotypic DST, a prototype version

Xpert MTB/XDR Cartridge displayed sensitivities:
 83.3% (ISONIAZID)
 88.4% (OFLOXACIN)
 96.2% (MOXIFLOXACIN)
 71.4% (KANAMYCIN)
 Further development will need to focus on
drugs suh as:

 Bedaquiline
 Pretomanid
 Linezolid
TRUENAAT (CHIP-BASED)
 2 step assay: 1 hour for MTB
& 1 hour for Rifampicin
Resistance detection
respetively.
 Based on Quantitative Real-
Time PCR (QRT-PCR).
 Battery operated and
amenable for placement in
peripheral settings.
 Molbio Diagnostics Pvt. Ltd.
(GOA)
 LINE PROBE ASSAYS (LPA)
 FL-LPA/SL-LPA performed directly on AFB-
Smear positive or indirectly on culture
isolates if smear is negative.

 Based on amplification by PCR and reverse

hybridization DNA strip technology.

 Results can be read visually or using

automated reader.

 Turn around time:1-3 days.

Reverse hyberdization
of amplified NAs to
specific DNApobes
bound on strips

Evaluation
Step 3: look at the results of WT and MUT band result
 ADVANTAGES DISADVANTAGES

Useful method to rule in TB and MDR Requires suitable facilities,adequate

TB. skilled laboratory personnel.

Detect mono resistance to isoniazid Not helpful in direct testing with –

and rifampicin. smear negative sputum,
Rapid turn around time-72 hours. paucibacillary TB,EPTB, Samples
with blood.
 FL-LPA:
 For rapid detection of drug resistance (Rifampicin,Isoniazid).

1. MTB-DRplus: RIFAMPICIN (rpoB gene)

ISONIAZID (katG & inhA)
2.BD MAX MDR-TB: Becton Dickinson MAX MDR-TB Assay

ISONIAZID & RIFAMPICIN Resistance.

 SL-LPA:
 For detection of drug resistance (FQL, SLIs)

1. MTB-DR sL: FQLs (gyrA gene)

SLIs (rrs gene)
TB-LAMP
[LOOP-MEDIATED ISOTHERMAL AMPLIFICATION]

 It is an isothermal PCR amplification technique that can

be performed in peripheral health care settings.

 Time taken< 1 hour.

 Result can be detected with naked eye under UV Light.
SEQUENCE BASED ASSAYS
 WHOLE GENOME SEQUENCING(WGS):

 WGS provides a comprehensive review of the entire

MTB genotype with a 96% concordance for culture
based sensitivity testing.

 It provides genotype sensitivity to most drugs required

for treatment of MDR-TB.

 Can provide individual patient centered DR-TB

treatment.
 NAAT and LPA may not detect clinically
relevant mutations outside the RRDR of rpoB
gene.

 For example-1491F mutation that has been

responsible for an outbreak of MDR-TB in
Eswatini and remains a grave public health
concern.

 METAGENOMICS(microbial cfDNA)
 TARGET MOLECULAR SCREENING
THANK YOU