MOLECULAR DIAGNOSIS OF TB

AND IGRA

PRESENTED BY :

Dr. Kiran N. PG Student

Chest & TuberculosisGovt. Medical College, Patiala

Molecular Diagnostics Why? Detection and Diagnosis- uncultivable or difficult to

culture- need for rapid diagnosis- inadequacy of phenotypic

methods (biochemical)- Prognosis and management- need for quantitative

information (bacterial load)- susceptibility testing (drug

resistance) without culture- Molecular resistance testing

METHODS OF DIAGNOSIS OF PULMONARY TUBERCULOSIS

1)DIRECT METHODS: Detects mycobacteria and its products

2)INDIRECT METHODS : Antigen and Antibody Detection

3)RADIO-DIAGNOSIS :CXR,CT AND MRIDIRECT METHODS1)Direct Microscopy -ZN stain, Kinyoun, Flurochrome.2)Culture -Traditional, Rapid methods. 3) Detection of DNA or RNA of mycobacterial origin i,e

(molecular methods) includes - PCR, LAMP, TAA / NAA, LCR, Fast Plaque.

INDIRECT METHODS1)Antibody detection : TB STAT-PAK ELISA Insta test TB

2) Antigen detection : TB MPB 64 patch test. Quantiferon-GOLD test.

3) Biochemical Assays : ADA Bromide Partition Gas Chromatography

MOLECULAR DIAGNOSIS OF TUBERCULOSIS

Rapid and sensitive tools for the diagnosis of tuberculosis are needed, due to the increased incidence of tuberculosis epidemics and the length of time required by classical diagnostic tests, especially among human immunodeficiency virus (HIV)-infected patients. In this context, the recent advances in cloning and characterization of M. tuberculosis genes has allowed the application of basic molecular biology techniques to the examination of clinical samples, such as sputum and bronchoalveolar lavage (BAL), for the molecular diagnosis of tuberculous infection. By using the polymerase chain reaction (PCR) for the amplification of mycobacterial nucleic acids and nonradiometric revelation techniques, the time required for the identification of mycobacteria has been considerably shortened (24-48 h), in comparison to the time required by microbiological tests

MOLECULAR TESTS FOR DETECTION OF NUCLEIC ACIDS

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 advance

• PCR is a technique that takes a small amount of a specific DNA sequence and amplifies for further testing.• It`s like a “molecular photocopier”.

PCR was invented by Kary Mullis in 1983 (Nobel Prize for Chemistry in 1993)

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR)

Essentially PCR is a way to make millions of identical copies of a specific DNA sequence , which may be a gene, or a part of a gene, or simply a stretch of nucleotides with a known DNA sequence, the function of which may be unknown.

A specimen that may contain the DNA sequence of interest is heated to denature double stranded DNA.

Specific synthetic oligonucleotide primers bind to the unique DNA sequences of interest and a heat stable DNA polymerase (Thermus aquaticus) extends the primer to create a complete & complimentary strand of DNA.

This process is repeated sequentially 25-40 times, thereby creating millions of copies of target sequence. The amplified sequence can then be detected by agarose gel electrophoresis.DNA sequence used include:

1) 65 Kd antigen (HSPs): Used earlier Heat shock protein believed to be distinct from other

bacterial HSPs. 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.

2) IS6110 : It is a transposon which are self replicating stretches of DNA. Function not known. 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 PCR can detect even a fraction of a bacilli.

Mycobacterium tuberculosisgenome

Ingredients of Polymerase Chain Reaction

• Primers: µM 0.1-0.5• Deoxy-nucleotides triphosphate (dNTPs):

µM 200-250nucleotides• Co-factors:

1. Cations: MgCl2 mM 1.5-6

2. Buffer pH 8.3-8.8• DNA polymerase: 0.5-2.5 U• Target DNA: 1 µg

Chromosome and Desoxyribo-Nucleic-Acid

Chromosom

Zelle

Zellkern

BasenpaareDNA-Doppelhelix

chromosome

cell

nucleus

base pairsDNAdouble helix

• Primers are target sequence specific oligonucleotides that serve as template for the DNA polymerase

• Forward and reverse primers flanking the target sequence allow both DNA strands to be copied simultaneously in both directions.

Primers

Three steps of PCR: Denaturation, annealing and extension

Role of PCR 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 Distinguish 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

Role in Extrapulmonary TB

Limited Role No comprehensive large series comparing

the yield of PCR with other available approaches has been published. But at present, it is valuable adjunct in the diagnosis of TBM, pleurisy, pericardial TB & other

condition in which yield of other tests are low

Disadvantages Very high degree of quality control

required. Variation from lab to lab remain

significant. In pts. on ATT, PCR should not be used as

an indicator of infectivity as this assay remains +ve for a greater time than do cultures

High false +ve results in patients previously treated with ATT in contacts of sputum +ve active cases.

High Cost

Real Time PCR replacing older Methods

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).

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.

ADVANTAGES:Due to its easy operation without

sophisticated equipment, it will be simple enough to use in:

Small-scale hospitals, Primary care facilities Clinical laboratorie in developing

countries.Difficulties : Sample preparation Nucleic acid extraction Cross-contamination

TMA / NAA Transcription Mediated Amplification (TMA)

/ Nucleic Acid Amplification (NAA). These techniques use chemical rather than

biological amplification to produce nucleic acid.

Test results within few hours. Currently used only for respiratory

specimens.

Nucleic acid amplification assays

NAA assays amplify M. tuberculosis-specific nucleic acid sequences using a nucleic acid probe.

The sensitivity of the NAA assays currently in commercial use is at least 80% in most studies

Require as few as bacilli from a given sample NAA assays are also quite specific for M.

tuberculosis, with specificity in the range of 98% to 99%.

NAAs- various types AMPLICOR M. TUBERCULOSIS assay

Amplified M.tuberculosis Direct (AMTD2) assay

LCx MTB assay, ABBOTT LCx probe system

BD ProbeTec energy transfer (ET) system (DTB)

INNO-LiPA RIF.TB assay

NAAs- various types

NAA- Limitations They are able to detect nucleic acids from

both living and dead organisms so in pts on ATT, PCR should not be used as an indicator of infectivity as this assay remains positive for a greater time, than do cultures

A major limitation of NAA tests is that they give no drug-susceptibility information.

NAA should always be performed in conjunction with microscopy and culture

DISADVANTAGES :

1) Poor sensitivity in smear –ve samples 2) Labour intensive 3) Highly trained personnel required & dedicated labour space because it requires atleast 3 separate rooms to avoid cross contamination. Hence suitable only for IRL.

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.

MDR TB MDR-TB is defined as resistance to isoniazid and rifampicin,

with or without resistance to other first line drugs (FLD). In the Global TB Report 2011, WHO estimated that among

the 1.5 million RNTCP-notified cases of pulmonary TB in India in 2010, approximately 64,000 cases of MDR TB could be diagnosed.

MDR TB is important because patients with this type of drug resistance respond extremely poorly to standard anti-TB treatment with first-line drugs. MDR TB requires relatively costly laboratory diagnosis and treatment for at least two-years with drugs that are expensive, toxic, and not particularly potent.

A case of MDR TB is about 20-40 times more expensive to manage than a case of drug-sensitive TB, and patient suffering is magnified.

MDR Suspect CriteriaCriteria A-• All features of new TB cases.

• Smear +ve previously treated cases who remain smear +ve at 4th

month onwards.

• All pulmonary TB cases who are contacts of known MDR TB case.

Criteria B – in addition to Criteria A :

• All smear +ve previously treated pulmonary TB cases at diagnosis.

• Any smear +ve follow up result in new or previously treated cases.

Criteria C – in addition to Criteria B :

• All smear –ve previously treated pulmonary TB cases at diagnosis.

• HIV TB co-infected cases at diagnosis.

Molecular methods for drug resistance

Rifampin (RIF) – Binds to β subunit of RNA polymerase (rpoB) – 96% of resistant Mtb isolates have mutations in 81-bp

region. – Four (4) mutations . 75% of resistant clinical isolates • Isoniazid (INH) . two genes – katG and inhA . 75-85% • Pyrazinamide . pncA . 70% • Streptomycin . rpsL . 65-75% • Ethambutol .embB . 70%

Causes of Drug Resistance in TB

Lack of understanding of why long term therapy with multiple drugs is necessary

Non-adherence to therapy by patients Incorrect drug prescribing by providers Poor quality drugs Erratic supply of drugs Malabsorption of drugs primarily due to

symptoms of HIV/AIDS

Development of drug resistance in M. tuberculosis

The mycobacterial cell is surrounded by a specialized, highly hydrophobic cell wall that results in decreased permeability to many antimicrobial agents.

Resistance of M tuberculosis to antimycobacterial drugs is the consequence of naturally occuring, spontaneous mutations in genes that encode either the target of the drug, or enzymes that are involved in drug activation.

Resistance-associated mutations have been described for all first-line drugs (isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin).

Development of multi-drug resistant tuberculosis

No single genetic alteration has yet been found that results in the MDR phenotype (defined as resistance at least to INH and RMP).

MDR develops by sequential acquisition and selection of mutations at different loci, usually because of inappropriate patient treatment.

Inappropriate treatment may lead to disease progression.

Disease progression will increase the bacterial load and the risk of naturally occurring mutations.

Because MDR strains are the result of cumulative mutations, growth of M tuberculosis can successfully be controlled in the host by concomitant treatment with more than one drug.

Thus, treatment regimens that consist of three to four drugs are used routinely to treat patients with tuberculosis.

Isoniazid resistance and katG INH is a pro-drug that requires activation

in INH-susceptible mycobacterial species.

the activation of INH results in a number of highly reactive compound that are capable of damaging the mycobacterial cell wall.

INH-resistant clinical isolates frequently loose their catalase-peroxidase activity (Middlebrook et al., 1954)

Association of this enzyme with INH activation was proven when the mycobacterial catalase-peroxidase gene (katG) was cloned and sequenced. (Zhang et al., 1992)

Mutations in this gene were found in 70-80% of high INH-resistant clinical isolates.

The most common mutation that was found was the Ser315Thr mutation.

Cytoplasm

Mycolic Acids

Free Wall Glycolipids and Proteins

Porin

Arabino- galactan

Lipoarabinomannan (LAM)

INH

PZA

RMP

Chromosomal DNA

RNA Polymerase (ß-subunit)

RNA Transcription

Short chain fatty acid precursors

Cel l W

a ll an d cyto p las mi c m

embra ne

Isoniazid resistance and virulence

The Ser315Thr mutation results in an enzyme without the ability to activate INH, but retains approximately 50% of its catalase-peroxidase activity.

This altered catalase-peroxidase provides high-level resistance to INH, while retaining a level of oxidative protection against host antibacterial radicals.

Isolates that carry other, mutations in katG are exhibiting varying levels of INH-resistance and catalase-peroxidase activity.

MTBPhagosome

with viable MTB

Lysosomes with free oxigene radicals

Phago-lysosomes with inactivated MTB

Isoniazid resistance and inhA INH blocks the synthesis of cell-

wall mycolic acids, the major components of the envelope of M tuberculosis.

One intracellular target of the drug is fatty-acid enoyl-acyl carrier protein reductase (InhA). (Basso et al., 1998)

This enzyme is involved in synthesis of mycolic acids.

Mutations in the promoter region of the gene (inhA) encoding this enzyme result in over-expression of the protein.

The over-expressed enzyme may counter-balance the effect of INH and will result in a low-level resistance to the drug.

Cytoplasm

Mycolic Acids

Free Wall Glycolipids and Proteins

Porin

Arabino- galactan

Lipoarabinomannan (LAM)

INH

PZA

RMP

Chromosomal DNA

RNA Polymerase (ß-subunit)

RNA Transcription

Short chain fatty acid precursors

Cel l W

all and cy top la smi c m

emb ran e

Rifampin resistance One of the main reasons

for treatment failure and fatal clinical outcome in tuberculosis patients is resistance to RMP.

RMP exhibits a significant early bactericidal effect on metabolically active M tuberculosis, and excellent late sterilizing action on semidormant organisms undergoing short bursts of metabolic activity.

While monoresistance to INH is common, monoresistance to RMP is rare.

RMP resistance occurs most often in strains that are also resistant to INH, thus, RMP resistance can be used as a surrogate marker for MDR.

RMP inhibits mycobacterial transcription by targeting DNA-dependent RNA polymerase.

Cytoplasm

Mycolic Acids

Free Wall Glycolipids and Proteins

Porin

Arabino- galactan

Lipoarabinomannan (LAM)

INH

PZA

RMP

Chromosomal DNA

RNA Polymerase (ß-subunit)

RNA Transcription

Short chain fatty acid precursors

Cel l W

all and cy top las mi c m

emb ran e

Rifampin resistance and rpoB

•Resistance to RMP is due to mutations in a well-defined, 81 base pair (27 codons) central region of the gene that encodes the β-subunit of RNA polymerase (rpoB). •More than 96% of the rifampin-resistant strains contain a mutation in this 81 bp region of rpoB.•The most common mutations (65–86%) alter either codon 526 or codon 531, and result in high-level resistance to RMP.•Alterations in other codons result in low-level resistance.•Rare mutations associated with rifampin resistance have also been found in the amino-terminal region of rpoB.

MOLECULAR METHODS OF DIAGNOSING DRUG RESISTANT

TB Phenotypic methods: in liquid/solid media Genotypic methods: Gene xpert/LPA

PHENOTYPIC METHODS1)Commercial methods Done on liquid or solid media Can be direct/indirect type. Indirect type has 3 variants: absolute

concentration/resistant ratio/proportion method Can be used for 1st line/2nd line DST

2)Non commercial methods Less expensive than commercial systems but are

prone to errors due to lack of standardization and local variations in methodology. These include

a)MODS (microscopic observation drug susceptibility) a microcolony method in liquid culture(middlebrook 7H9 broth) based on inoculation of specimens into drug-free and drug-containing media, followed by microscopic examination of early growth Recommended as direct or indirect tests for rapid screening of patients suspected of having MDR-TB.

b)COLORIMETRIC METHODS Indirect testing methods based on the reduction of a coloured indicator added to liquid culture medium on a microtitre plate after exposure of M. Tuberculosis strains to anti-TB drugs in vitro. Recommended as indirect tests on M. tuberculosis isolates from patients suspected of having MDR-TB, although the time to detection of MDR is not faster (but less expensive) than conventional DST methods with commercial liquid culture or molecular LPA. The indicators that have been used to date include tetrazolium salts (XTT and MTT), Alamar blue and resazurin.

A direct or indirect method on solid culture based on the ability of M.tuberculosis to reduce nitrate, which is detected by a colour reaction Recommended as direct or indirect tests for screening patients suspected of having MDRTB, although the time to detection of MDR in indirect application is not faster than conventional DST methods with liquid culture.

c)NITRATE REDUCTION ASSAY (NRA)

GENOTYPIC METHODS

LPA Gene

Xpert /RIF

LPA(Line Probe Assay)It is a genotypic method use PCR and reverse hybridization with specific oligonucleotide probes fixed to nitrocellulose strips in parallel lines therefore often reffered to as “strip tests”. which detects resistance to

Both H&R Sensitivity for R is 97%, H is 90% Specificity for R& H is 99% Results within 48 hours

DifferencesInnoLiPA Rif

• It targets 16s - 23s rRNA gene space region.

• It detects only R resistance.

Hain GenotType MTBDRplus

• It targets 23s rRNA gene space region.

• It detects both R and H resistance.

TB Molecular Identification

ADVANTAGES of molecular testing:

Rapid results

Specific information

Can be done in presence of contaminants

Less biohazard risk involved

DISADVANTAGES of molecular

testing:

Expensive

Dedicated equipment

Technical expertise required

AVAILABLE SYSTEMS (Commercial):

Hain GenotType MTBDRplus

InnoLiPA Rif

Rapid molecular detection of INH and RMP resistance: Line – Probe Assay Methodology

1. DNA isolated from processed original specimen or from cultured bacterial cells

2. Multiplexed amplification (PCR) of mycobacterial DNA using biotin labeled primers

3. Denaturation of the amplified DNA products into single-strands

4. Hybridization of the denatured DNA to probes on a membrane strip

5. Enzyme-mediated detection of bands where DNA products have bound to strip

Polymerase Chain Reaction

MTBDR plus strip for Identification of MTB complex

and resistance to RMP and/or INH

Rifampicin resistance: rpoB gene

51 1 51 3 51 6 51 8 52 2 52 6 53 1 53 3

r po B W T2 r po B W T4 r po B W T6r p o B W T8r po B W T7

r po B M U T 1 ( D 5 1 6 V )r po B M U T3 (S 531L )

r po B M U T 2 B ( H 5 2 6 D )

51 4 51 5

r po B W T1 r po B W T3 r p o B W T5

50 950 850 5

• rpoB wild type probes: WT 1 to WT 8• rpoB mutation-specific probes: MUT D516V, H526Y, H526D, S531L

Detection of mutations:• missing of wildtype signals• presence of common mutation-specific signals

High-level Isoniazid resistance: katG gene

Mutations in katG and the corresponding wild type and mutation probes

missing wild type probe analyzed codon mutation probe mutation

katG WT 315 katG MUT1 S315T1katG MUT2 S315T2

High-level Isoniazid resistance: katG gene

Mutations in katG and the corresponding wild type and mutation probes

missing wild type probe analyzed codon mutation probe mutation

katG WT 315 katG MUT1 S315T1katG MUT2 S315T2

High-level Isoniazid resistance: katG gene

Mutations in katG and the corresponding wild type and mutation probes

missing wild type probe analyzed codon mutation probe mutation

katG WT 315 katG MUT1 S315T1katG MUT2 S315T2

Low-level Isoniazid resistance: inhA gene

Mutations in the inhA promotor region and the corresponding wild type and mutation probes

Xpert MTB/RIF assay (CBNAAT-cartridge based

NAA test) It is an automated PCR diagnostic

test that can detect presence of M.tb & resistance to RIFampicin(by detecting mutation in 81bp region)

It is automatic,fast & sensitive Accurate diagnosis is obtained in

1hr 45 mins by adding a reagent to the sputum sample & 15mins later its pippeted into a cartridge that is inserted into the diagnostic instrument

Xpert MTB/RIF Dual PCR reactions—

sample-processing PCR is followed by hemi-processing PCR —increase the test’s sensitivity and specificity: according to the results published in NEJM, the PCR test was 98.2% sensitive in patients with smear-positive, culture-positive TB. And, because it is automated, there is little technical training needed to administer the test.

Other molecular methods include:3) DNA microarrays Based on the principle of hybridization Detect Rifampicin resistance only Analysis large amounts of DNA sequences4)Molecular beacons They are nucleic acid hybridization probes d desinged to bind to

target DNA sequences in regions such as rpoB ,where resistance mutations are known to occur.

• Sensitive enough to detect 2 bacilli, • Results in 3 hours

5)Single-strand conformation polymorphism (SSCP)

6)Fluorescence resonance energy transfer (FRET) probes

7)Flow cytometry

CONCLUSION:LPA is applicable on sputum+ve specimens only.Gene Xpert cant monitor progress of treatment. HENCE PHENOTYPIC CULTURE REMAINS THE GOLD STANDARD

Interferon-gamma release assays for latent tuberculosis

infection They are surrogate markers of Mycobacterium tuberculosis infection and indicate a cellular immune response to M. tuberculosis.

IGRAs cannot distinguish between latent infection and active tuberculosis (TB) disease, and should not be used for diagnosis of active TB, which is a microbiological diagnosis.

A positive IGRA result may not necessarily indicate active TB, and a negative IGRA result may not rule out active TB.

IGRAs are not affected by Bacille Calmette-Guérin (BCG) vaccination status, IGRAs are useful for evaluation of LTBI in BCG-vaccinated individuals, particularly in settings where BCG vaccination is administered after infancy or multiple (booster) BCG vaccinations are given.

IGRAs appear to be unaffected by most infections with environmental nontuberculous mycobacteria

Blood Assays for M. tuberculosis

QuantiFERON®-TB Gold In-Tube (Cellestis Ltd, Victoria, Australia) Measures Interferon-gamma (IFN-y)

T-SPOT.TB (Oxford Immunotec Ltd, Oxford, UK) Measures peripheral blood

mononuclear cells that produce IFN-γ

QuantiFERON-TB Gold In-Tube assay (Cellestis Ltd, Australia)

The QuantiFERON-TB Gold In-Tube assay (QFT-GIT), which has replaced the QuantiFERON-TB Gold assay, detects the level of IFN-γ produced in response to the M. tuberculosis antigens ESAT-6, CFP-10, and TB7.7, and uses the enzyme-linked immunosorbent assay (ELISA) detection method. This is an indirect measure of the presence of M.tuberculosis specific T-cells.

T-SPOT.TB assay (Oxford Immunotec, UK)

The T-SPOT.TB measures the number of IFN-γ producing T-cells in response to the M. tuberculosis antigens ESAT- 6 and CFP-10, and is based on the enzyme-linked immunosorbent spot (ELISPOT) assay.

TST QFT-GOLD InTube T-Spot.TBAdministration In vivo (intradermal) Ex vivo ELISA-based Ex-vivo Elispot

based

Antigens PPD-S or RT-23 ESAT-6 + CFP-10 +/- TB 7.7

ESAT-6 + CFP-10

Standardized Mostly Yes Yes

Units of measurement Millimetres of induration

International units of IFN-G

IFN-G spot-forming cells(SFC)

Definition of positive test

5,10,15 mm Patient’s IFN-G>0.35 suIU/ml (after subtracting IFN-G response in nil control)

>6 SFC in the antigen wells,with 250,000 cells/well and atleast double negative well

Inderminate If anergy (rarely tested )

Poor response to mitogen (<0.5 IU/ml in positive control) or high background response (>8.0 IU/ml in nil well)

Poor response to mitogen (<20 SFC in positive control well) or high background (>10 SFC in negative well)

Time to result 48-72 hrs 16-24 hrs (but longer if run in batches)

16-24 hrs (but longer if run in batches)

Cost per test $12.73 $41 $85

CHARACTERISTICS OF THREE TESTS FOR LTBI

COMPARISION OF TST AND IGRAs TST IGRA

Estimated sensitivity in patients with active TB 75-90% 75-95%

Estimated specificity in healthy individuals with no known TB disease or exposure

70-95% 90-100%

Cross reactivity with BCG Yes Less likely

Cross reactivity with NTM Yes Less likely but limited evidence

Association between test-positivity and subsequent risk of active TB during follow- up

Moderate to strong positive association

Insufficient evidence

Correlation with Mycobacterium tuberculosis exposure

Yes Yes(better than TST)

Benefits of treating test-positives based on RCTs Yes No evidence

Reliability and reproducibility Moderate and variable

Limited evidence but appears high

Boosting phenomenom Yes No

Potential for conversions and reversions Yes Insufficient evidence

Adverse reactions Rare Rare

Material costs Low High

Patient visits Two One

Laboratory infrastructure required No yes

Time to obtain results 2 to 3 days 1 to 2 days

Trained personnel required Yes yes