recent advances in diagnosis of Tuberculosis

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Introduction :

Introduction Inexpensive, rapid, and reliable methods of detecting infection Crucial to the control of tuberculosis Novel drug susceptibility testing Early growth More timely diagnosis and Drug susceptibility testing


MICROSCOPY TO PREDICT DRUG RESISTANCE (VITAL STAINING) For > 100 yrs Sputum Microscopy – cornerstone in diagnosis Most TB patients worldwide diagnosed by Smear microscopy Hence Microscopy + Prediction of drug resistance – VITAL STAINING First developed for Mycobacterium leprae in 1982 Can be applied to MTB Fluorescein diacetate (FDA) is hydrolyzed intracellularly to Fluorescein - rapidly accumulates in viable bacilli – detected under UV illumination with LED

Mtb Vital staining:

Mtb Vital staining Serial Sputum examination To follow response of Patient to RX Persistent MTB viability - Drug resistance Not specific Useful in resource poor settings Hamid Salim A, Aung KJ, et al, Int J Tuberc Lung Dis, 2006;10(11):1248–54. [PubMed: 17131784]

FDA Staining – Method :

FDA Staining – Method Flood the smear with FDA (0.5 mg /ml) 30 minutes at 37 ºC incubator Rinse with water 1% acid alcohol 3 minutes (de-colorization) Rinse with water 10 minutes 5% watery phenol (safety step) Rinse with water Dry the smear (30 min -1 hour) in dark Read as soon as possible FDA Dead bacilli Live bacilli

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Feasibility Simple procedure Standard microscope equipped with LED cassette required Limitations: -20°C freezer (storage of FDA stock solution) Fading of fluorescein signal Scanty-loaded AFB-sputum may be miss-read as “fluorescein-negative” Resource poor setting for prediction of Drug resistance Operational aspects of FDA vital staining method in a peripheral smear microscopy

Drug susceptibility testing:

Drug susceptibility testing


PHENOTYPIC METHODS Absolute concentration method Resistance ratio method Proportion method BACTEC 460 MGIT 960 MB / Bac T system and ESP II

Recently developed phenotypic methods :

Recently developed phenotypic methods EPSILOMETER TEST (E Test) COLORIMETRIC ASSAYS Micro well Alamar Blue assay Microplate tetrazolium reduction assay Nitrate reductase assay MYCOLIC ACID INDEX SUSCEPTIBILITY TESTING (MAI) MICRO COLONY DETECTION (MODS) PHAGE BASED ASSAYS Phage based assay Luciferase reporter phage assay

Genotypic methods :

Genotypic methods Automated DNA sequencing PCR SSCP PCR HDF LiPA (solid phase hybridization assay) Miscellaneous genotypic methods In House Assays




SAMPLE PREPARATION FOR PHENOTYPIC METHODS Sputum decontamination and concentration is must Requires biosafe centrifuge and appropriate infection control measures Direct DST To be performed in BSL2 settings , requires larger innoculum Indirect DST require BSL 3 containment

E-Test - Indirect DST:

E-Test - Indirect DST The E-test use with M. tuberculosis was first described in 1994. Commercially available as AB BIODISK Based on determination of drug susceptibility using strip containing gradients of impregnated antibiotics High rate of false resistance compared with BACTEC or LJ proportionate methods

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Etest results for a clinical isolate ofM. tuberculosis after 10 days of incubation. Hazbón M H et al. J. Clin. Microbiol. 2000;38:4599-4603 RIF STR INH Modest performance High cost BSL 3 facility required .

Non commercial DST tests for screening patients at risk for MDRTB :

Non commercial DST tests for screening patients at risk for MDRTB CRI, NRA MODS, TLA & Phage based Assays WHO POLICY Direct Testing – MODS and NRA Indirect DST – CRI, MODS and NRA


COLORIMETRIC DST METHODS Low-tech, low cost approach to MTB growth detection Oxidation-reduction indicator changes color in response to the metabolic products associated with MTB growth, or Nitrate reduction by replicating MTB - revealed by an added indicator When isolates are cultured in a range of concentrations of anti-TB drugs - gives MIC (min conc. where color change is inhibited)


DIFFERENT COLORIMETRIC ASSAYS MABA (Microplate Alamar blue assay) Tetrazolium microplate assay (TEMA) Nitrate reductase Assay (NRA)

MABA (microplate Alamar blue assay):

MABA (microplate Alamar blue assay) Alamar Blue, a resazurin change from blue (oxidized state) to pink in the presence of bacterial growth Gives reliable first and second line DST by MICs

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Outer perimeter wells were filled with sterile water to avoid dehydration A standard bacterial suspension = No. 1 McFarland standard was prepared and diluted 1:20 in 7H9 broth Additional control wells consisted of bacteria only (B) and medium only (M). Antibiotic concentrations are prepared directly in the wells After 5 days’ incubation at 37C, the indicator (20 µ l of Alamar Blue and 12 µ l of sterile 10% Tween 80) was added to a drug-free growth control wells Plates were re-incubated for 24 h. If the control well turned pink, all other wells will receive the indicator. After a further 24 h of incubation, the colours of all wells were recorded

Tetrazolium microplate assay (TEMA):

Tetrazolium microplate assay (TEMA) Oxidation reduction of dye tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Drug resistance indicates changing color from yellow to violet by replicating bacteria Performs well in Microplate format for both first and second line DST .

Tetrazolium microplate assay (TEMA) – Innoculum preparation :

Tetrazolium microplate assay (TEMA) – Innoculum preparation Pellet obtained is resuspended in distilled water Sample processed by modified petroff’s method and centrifuged Pellet + supplemented 7H9 Middlebrook broth Pellet + unsupplemented 7H9 Middlebrook broth (CONTROL)

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100 μL of Middlebrook 7H9 broth + Bacterial suspension The antibiotics were serially diluted twofold in consecutive columns The plates were incubated at 37°C for 5 days The wells in column 11 served as inoculum-growth control with no drugs On day 5, 50 μl of the tetrazolium dye was added to well A11 and the plate was then incubated at 37°C for 24 h A change in color from yellow to purple indicated growth of bacteria and the MICs was interpreted visually.

Nitrate Reductase Assay :

Nitrate Reductase Assay Fresh subculture of M. tuberculosis grown on LJ medium was taken and vortexed in 3ml of phosphate buffer saline (PBS, p H 7.4) 0.2 ml of suspension was inoculated into the tubes containing LJ medium with potassium nitrate (KNO3) and the antitubercular drugs Tubes were incubated at 37°C for 14 days 0.5 ml of a mixture of three reagents (25 μl of concentrated HCl + 50 μl of 2% sulphanilamide & 50 μl of 1% n-1-napthyl-ethylenediamine dihydrochloride) was added – observe change in colour

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RMP INH SM EMB GC RMP resistant strain Susceptible strain


NEW COLORIMETRIC METHOD A novel, simplified colorimetric culture method on TK Medium™ (Salubris, Inc., MA, USA). Metabolic activity of dividing mycobacteria is detected by a change in the color ( red to yellow), Contamination ( red to green )

CRI Methods review:

CRI Methods review CRI methods are indirect tests, The time to diagnosis of MDR not faster than conventional phenotypic DST or genotypic testing with line-probe assays. Yet, CRI methods are highly sensitive Manipulation of cultures – aerosol formation Hence Containment with BSL 3 must

Mycolic acid index susceptibility testing (MAI):

Mycolic acid index susceptibility testing (MAI) Mycolic acids – α alkyl β hydroxy acids of high mol wt 1986 , W. R. Butler – Quantification of p- Bromophenacyl derivatives of mycolic acids using HPLC 1995, K Josh et al – diverse fluorescent coumarin derivatives of mycolic acids – diff mycobacterial species E. Garzagonzalez – described exponential relationship between total area of mycolic acid (TAMA) peaks of culture of mycobacterium tb and viable count

Mycolic acid index (MAI):

Mycolic acid index (MAI) Rate of mycolic acid increase during incubation in presence of a drug and the mycolic acid increase in the absence of a drug Total area under the mycolic acid (TAMA) chromatographic peaks of a culture of M. tuberculosis ∞ log CFU per milliliter Max time required 5 days

Mycolic acid index (MAI)- inoculum preparation :

Growth from LJ medium is homogenized with SDW & Glass beads Opacity adjusted to McFarland standard 0.5 100 µl of this suspension is added to Two tubes – 1ml of 7H9 broth with out antibiotics One tube - broth plus 0.2mg of INH/ml One tube - broth plus 2.0 mg of RIF/ml Mycolic acid index (MAI)- inoculum preparation

Mycolic acid index (MAI)- incubation and result :

The drug-containing tubes & the drug-free tubes were incubated for 5 days at 37°C Afterwards, mycolic acid analyses is carried out by HPLC using fluorescence detection. Mycolic acid index (MAI)- incubation and result

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Mycolic acid pattern of clinical isolate from drug free tube before incubation Mycolic acid pattern of clinical isolate after 5 days incubation Mycolic acid pattern of clinical isolate in the presence of INH Mycolic acid pattern of clinical isolate in the presence of RIF

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Mycolic acid pattern of clinical isolate from drug free tube before incubation Mycolic acid pattern of clinical isolate after 5 days incubation Mycolic acid pattern of clinical isolate in the presence of INH Mycolic acid pattern of clinical isolate in the presence of RIF

Microscopically observed drug susceptibility testing (MODS):

Microscopically observed drug susceptibility testing (MODS) MODS assay, directly from sputum, relies on three principles: MTB grows faster in liquid medium characteristic cord formation can be visualized microscopically in liquid medium at an early stage the incorporation of drugs permits rapid and direct drug-susceptibility testing concomitantly with the detection of bacterial growth.

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24-well tissue-culture plates (each well containing decontaminant, 7H9broth, OADC and PANTA supplements) For each sample, 4 wells were used: 2 drug free controls and One well for rifampicin and One well for INH. 100µl diluted bacterial samples were inoculated Plates were sealed with polyethylene tape, incubated at 37°C with 5% CO2, observed with an inverted microscope at 100x Positive cultures were identified by cord formation

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Day 23 100x Day 5 100x Day 7 100x Day 9 100x Day 21 100x


THIN LAYER AGAR FOR MTB DETECTION Employs observation of micro colonies on a thin layer of 7H11 Agar plate and is used for drug sensitivity testing Sensitivity of TLA compares favorably with LJ Time to detection is significantly shorter at 7-11 days Less expensive than conventional method Good low cost alternative for resource poor countries

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The sample is inoculated on a plate containing Middlebrook 7H11 and Middlebrook 7H11 enriched with PNB (para-nitrobenzoic acid) Mtb complex grows on 7H11 agar but not on 7H11 + PNB Incorporation of drug into the medium and subsequent growth of organism indicates resistance Plates are observed using conventional microscope (10x) up to six weeks Growth of M. tuberculosis complex looks like small cords, further incubation – increase of cords formation THIN LAYER AGAR

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5 days incubation at 37°C (10X) 6 days incubation at 37°C (10X) 8 days incubation at 37°C (10X) 10 days incubation at 37°C (10X) 11 days incubation at 37°C (10X) 13 days incubation at 37°C (10X)

Phage based assays:

Phage based assays In the presence of a drug, the phages are only able to replicate if a drug-resistant strain is present. Visualization of the amplified phages can be performed either by counting the plaques or by determination of the light emission.

Luciferase reporter Phage Assay :

Luciferase reporter Phage Assay Viable mycobacteria are infected with recombinant mycobacteriophages expressing firefly luciferase gene. Easy detectable signals seen after few minutes after infection with M. tb Drug resistant strains are unaffected by drug and produce light levels equivalent to the untreated strains




SAMPLE PREPARATION FOR PHENOTYPIC METHODS Biosafety is not a concern for genotypic tests (MTB is heat-killed at the outset), But the issue is DNA cross-contamination Concentration, DNA extraction and Amplification processes Thus separate clean rooms dedicated purely to DNA extraction and amplification, and special attention to procedural care and detail, are needed.

Genotypic methods :

Genotypic methods For rapid detection of MRD TB Not plasmid mediated MDR TB usually results from accumulation of accumulation of individual target genes Faster turnaround – one working day

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Drug Gene Function Isoniazid kat G Catalase peroxidase inh A Enoyl-acyl carrier protein reductase ahp C Alkyl-hydroperoxide reductase kas A Ketoacyl acyl carrier protein synthetase Rifampicin rpo B β-subunit of the RNA polymerase Pyrazinamide pnc A Pyrazinamidase Streptomycin rps L Ribosomal S12 protein rrs 16S rRNA Amikacin/kanamycin rrs 16S rRNA Capreomycin rrs 16S rRNA tly A rRNA methyltransferase Fluorochinolone gyr A, gyr B DNA gyrase Ethambutol emb CAB Arabinosyl transferase

Automated DNA Sequencing :

Automated DNA Sequencing Provide highest level of information DNA sequencing of PCR amplified products – GOLD STANDARD Performed both manually and automatically DNA sequencing is used for characterization of mutation responsible for drug resistance Used mainly for drugs like Rifampicin, Isoniazid Streptomycin and Ciprofloxacin CUMBERSOME & EXPENSIVE EQUIPMENTNEEDED

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Single stranded DNA is amplified in the presence of fluorescently labelled ddNTPs that serve to terminate the reaction and label all the fragments of DNA produced. The fragments of DNA are then separated via poly acrylamide gel electrophoresis and the sequence read using a laser beam and computer.

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PCR SSCP (single strand confirmation polymorphism) Other genotyping methods PCR heteroduplex formation Hybridisation assays DNA Microarrays PCR restriction polymorphism analysis and REAL TIME PCR Techniques


REAL TIME PCR Advantage – running in closed system Minimal contamination Xpert MTB / RIF, Cepheid Simultaneous detection of both MTB and rifampicin resistance, ( MDR strains ) Unprecedented sensitivity for detecting MTB — even in smear negative, culture positive specimens Results in two hours; requires no instrumentation other than the GeneXpert® System

Cepheid GeneXpert MTB/RIF Boehme et al. 2010 NEJM :

Cepheid GeneXpert MTB/RIF Boehme et al. 2010 NEJM Automated RT-PCR Simple 1-step specimen preparation Results in 2 HOURS!! Sensitivity for diagnosis 99% in smear + 80% in smear - / culture + Rifampicin resistance 95% sensitive 98% specific

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DNA extraction from isolates or specimens Multiplex PCR with biotinylated primers – specific resistant gene Double stranded PCR products denatured Hybridized to immobilized membrane bound probes Annealed biotynylated oligonucleotide – detected by Enzyme mediated color reaction LINE PROBE ASSAY

In House Assays:

In House Assays In house tests targeting rpoB, gyrA and pnc A fpr detection of Pyrazinamide resistance Good results Further evaluation needed INNO-LiPA Rif, TB assay GenoType MTBDR plus

INNO-LiPA Rif, TB assay :

INNO-LiPA Rif, TB assay

Limitations of molecular Assays :

Limitations of molecular Assays Cannot target all possible genes or mechanisms involved ? Reliable identification of heteroresistance Better detected by classical susceptibility tests

Control of MDR / XDR TB:

Control of MDR / XDR TB Massive scaling-up of culture and DST capacity Expanded use of novel and rapid assays for drug resistance

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