Expert Commentary - 5 year review (2011)

TUBERCULOSIS ASSAYS

• Recent developments in the field of TB diagnostics

• No point-of-care tests • No tests - universally applicable to all

patients • Quick identification - TB control improves

INTRODUCTION

PLAN• The WHO Stop TB Strategy & Global Plan

objectives are 1. TB diagnostics - universal access to quality

diagnostics 2. Detection : 84% of cases by 2015 3. Updated goals: 2011–2015 ------> increased

diagnosis of active TB at point-of-care level 4. Screening for MDR & (XDR) TB

• Prospective study in South Africa • 17% of 367 TB cases dx on smear results

did not start tx • Incomplete sputum sample collection • Problems of sample transport • Poor record-keeping of samples & results

• Key to effectively combat TB remains comprehensive case finding & reporting

SPECIAL TARGET GROUPS

• HIV co -infection

• Women

• Children

HIV co-infection

• Clinical &radiological presentation • Symptoms : nonspecific or subclinical • Presence of co-infections • 63% of adult cases with PTB remained undiagnosed in an

efficient DOTS program • Passive case finding in HIV-infected individuals only

identified 33% of the smear-positive TB cases

Women

• Social welfare • Timely TB screening, dx & tx • Prevent intra-uterine complications in

pregnancy • Prevent perinatal & childhood TB

CHILDREN

• First 4 years of life • Risk of progression to active disease is high • Acute TB may develops within short interval • Not often seen as a priority group in endemic

areas/ in development of new diagnostics • Clinical signs of pulmonary & extrapulmonary dx

can present in combination • Microbiologically difficult to diagnose:

paucibacillary disease • Child with TB = Failure of system

Clinical Diagnostic Methods

• Clinical examination : unremarkable in early phases

• ? Reliability of classic TB symptoms • Cough, night sweats, loss of weight and fatigue

are reported in poor communities • HIV-coinfection : atypically or no symptoms • Timely dx of paucibacillary disease

Radiology

• CXR - suboptimal specificity & sensitivity • Clinical exam vs patient's radiographic picture • Common radiological findings : subject to micro

confirmation • HIV coinfection: degree of immune suppression • CT scans : higher sensitivity • Not widely accessible & requires specialist

services

Bacteriological methods

• Majority of Pxs can be diagnosed with the 1st sputum

• Increase in average sensitivity by examining a 3rd sputum specimen is low

• Patient benefit if 2 specimens can be collected on the same day

NO OF SPUTUM SAMPLES

• 20 studies using culture as ref standard

• Average sensitivity of 1st specimen :54%

• Average increase in sensitivity of 2nd specimen : 11%

• Average increase in sensitivity of 3rd specimen : 3%.

REVIEW

WHO POLICY In 2007, WHO revised the definition of a sputum smear-

positive pulmonary TB case to be based upon the presence of at least one AFB in at least one sputum

sample

Minimum number of sputum specimens to be examined was reduced from 3 to 2

Sputum Smear Microscopy • ZN staining : most widely used & cost-effective

• Detection rate : 60% in smear+ individuals

• In settings with high incidence of HIV infx : 20–40%

• Immuno-compromised pxs : lower bacterial loads

• AFB detection : 104 bacilli/ml of specimen

• Cannot differentiate between MTB vs NTM disease

• Up to 30% of pxs : unable to produce sputum

IMPROVEMENT IN MICROSCOPY

• Centrifugation, sedimentation, bleach• Bleach reported to increase sensitivity of

smear microscopy• Concentration step ffg sputum processing :

33% improvement over direct microscopy

REVIEW• 83 studies • Centrifugation with any of several

chemical methods is more sensitive • Overnight sedimentation preceded by

chemical processing is more sensitive • Specificity is similar • Insufficient data : HIV + pxs • Operational studies still needed

Fluorescence microscopy

• More sensitive for dx of PTB than conventional microscopy

• Specificity of FM for detection of AFB in sputum is high & similar to that of conventional microscopy

• Fluorochrome-stained smears take less time to examine than smears stained with ZN

REVIEW• 45 studies included

• Sensitivity of conventional microscopy (ZN) : 32% - 94%

• Sensitivity of FM : 52% - 97%

• FM was on average 10% more sensitive than conventional microscopy

• Specificity of FM high (98%) & similar to conventional microscopy

• There was insufficient evidence to determine the value of FM in HIV-infected individuals

• Concerning work effort • Large study involving 23,427 specimens • FM : 1 minute • conventional microscopy :4 minutes • FM: higher sensitivity & equivalent specificity

compared with conventional microscopy • FM dependent on expensive, complex

equipment & mercury vapour lamps • Inappropriate for use outside of reference

laboratories • New low-cost simple FM systems based on

Light-Emitting Diodes (LEDs)

LED MICROSCOPY

• Does not need expensive mercury vapour lamp or dark room

• In 2009, WHO recommended that FM be replaced by LED microscopy in all settings

• LED microscopy be phased in as an alternative for ZN microscopy in both high & low-volume labs

REVIEW• 12 studies included, culture as reference (8 studies)

• LED sensitivity : 67% - 96%

• Specificity : 89% - 100%

• In meta-analysis, pooled sensitivity was 84% & pooled specificity 98%

• LED microscopy was 6% more sensitive - with no loss in specificity, compared with ZN

• In comparison with ZN , LED microscopy showed similar gains in time for reading as FM

• Half the time required for microscopic smear examination

Phenotypic Culture & Drug Susceptibility Testing

• Culture : gold standard • 10–100 M. tuberculosis bacilli/mL of

specimen • MGIT reduces time to + result • Increases sensitivity (up to 10% higher)

over the traditional based culture methods • Other benefits • Culture-based DST - important

• DST : clinical specimen onto media • Inoculum from pure culture - Indirect method • Decontaminated clinical specimen- Direct

method • Direct method - reduces the turnaround time to

1–3 weeks • When performed in liquid media : increased risk

of contam & higher rate of NTM recovery • Indirect proportion method on agar media

remains gold standard for phenotypic-based DST • Method is reliable for 1ST line drugs INH& RIF but

inconsistence common in case of EMB resistance

• Significant delay with culture-based dx & DST

IMPROVEMENTS TO REDUCE DELAY-PHENOTYPIC DST METHODS • Microscopic detection of early mycobacterial

growth (thin-layer agar) • Microscopic-observation drug-susceptibility

assay • Colorimetric assays : alamar blue/resazurin

(4,5-dimethylthiazol-2-yl) ; 2,5-diphenyltetrazolium bromide & nitrate reductase assays

• Phage amplification assays

THIN LAYER AGAR

• Based on microscopic detection of early growth• Detect growth within 9–14 days • Initial ID based on colony morphology• Sample inoculated on Middlebrook 7H11 &

Middlebrook 7H11 enriched with PNB (para-nitrobenzoic acid)

• Growth compared• Growth of MTB complex on TLA in 1ST few days:

small cords• After few days : colonies are bigger in size & are

constituted by cords

MODS• 1. Growth faster in liquid medium• 2. Cord formation can be visualized

microscopically in liquid medium at early stage

• 3. Incorporation of drugs permit rapid & direct drug-susceptibility testing including detection of bacterial

• Disadvantage: Requires inverted microscope

REVIEW: TLA & MODS• Meta-analysis: 12 studies (9 for MODS & 3 for TLA

assays)

• MODS: Detection of RIF resistance : pooled sensitivity : 98% & pooled specificity : 99%

• Detection of INH resistance, pooled sensitivity was slightly lower depending on drug concentration used

• TLA: pooled sensitivity & specificity estimates RIF resistance (3 studies) & INH (2 studies) were 100%

• Time for results : 10 days for MODS • 11 days for TLA assay

WHO POLICY

• In 2009, WHO recommended selective noncommercial methods be used as an interim solution while advanced systems were being developed

• Concerns about mycobacterial speciation & biosafety were addressed

• Current evidence on TLA assays was insufficient to recommend their use.

COLORIMETRIC ASSAYS

• Growth indicators used- especially RIF & INH resistance

• Faster than conventional DST

• Reduction of coloured substrate, indicator added to culture medium after MTB has been exposed to different antibiotics

• Resistance detected by change in colour of the indicator, which is directly proportional to no. of viable mycobacteria in the medium.

• 7–10 Different indicators have been evaluated

• Tetrazolium salts: XTT [2,3-bis- (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] and MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium- bromide] and the redox indicators Alamar blue and resazurin

REVIEW• Meta-analysis: Highly sensitive & specific for

rapid detection of RIF&INH resistance in culture isolates

• Majority of studies : sensitivity 95%• RAPID TAT : between 7 & 14 days

• Performed on culture isolates: primary isolation is needed to do the test requiring a minimum of 2–6 extra weeks

• Useful : MDR-TB is strongly suspected• Cost-effectiveness of the colorimetric

assays over conventional method• Studies needed to measure performance

of test in countries with high prevalence of MDR-TB

• Accuracy of colorimetric methods applied directly to clinical specimens

• It will save a great deal of time if tests for MDR-TB can be performed directly on sputum samples

NITRATE REDUCTASE ASSAY

• Based on the capacity of MTB to reduce nitrate to nitrite

• Detected by adding a chemical reagent to the culture medium

• MTB growth in presence of antibiotic

• Ability to reduce nitrate measured after 10 days of incubation

• Resistant strains will reduce the nitrate : pink-red colour change

REVIEW• Multi -centered study : performance of NRA to detect

resistance to 1st line drugs

• Test performed very well for isoniazid, rifampicin & ethambutol with accuracy between 96.6 & 98%

• Lower values for streptomycin

• Easily implemented in countries with limited laboratory facilities

• Main advantage is it uses the same format & culture medium as in conventional method

PHAGE AMPLIFICATION ASSAYS• Utilize bacteriophages

• Two main approaches used to detect MTB

• (1) amplification of phages after infection of MTB ffg by detection of progeny phages using Sensor cells (plaque formation)

• (2) detection of light produced by luciferase reporter phages (LRP) after infection of live MTB

• Phage amplification methods more evaluated than LRP

• Phage-based assays are available as commercial kits

• FASTPlaque-TB & PhageTek MB & in-house assays

• In-house assays use either amplification technology or LRPs

• Some phage-based assays specifically designed to rapidly detect RIF resistance : FASTPlaque-TB-MDRi (FASTPlaque-TB-RIF)

• Newer kits being developed

REVIEW• Current evidence largely restricted former

for RIF resistance detection in culture isolates

• Relatively high sensitivity• Specificity lower & more variable : can

result in over-dx of MDR-TB in low prevalence

• Evidence lacking when directly applied to sputum specimens

• Better standardization of the assay needed

HOWEVER...• Not capable of producing results within a wk

• Expensive reagents

• Bio safety facilities

• Further speciation

• Genetic markers specific to MTB genome

GENOTYPIC METHODS

•Drug-resistant TB : various mechanisms •SNPs in specific genes of MTB : targeted by anti-TB drug

• ID of these SNPs forms basis of genotypic DST

•Genotypic-based dx: more data points •Phenotype not dependant on culture •More specific & Rapid

Sequencing

• DNA sequencing : gold standard • Reveals the complete genetic profile of

the region targeted by the anti-TB drug • Can be used for species ID • Resistance -causing mutations • Screen for novel SNPs that may be

associated with drug resistance • Major drawbacks : cost & skill

Commercial Genotypic-based Assays

• In-house PCR & commercial-based methods are available

• Genotypic-based assays currently endorsed by WHO for use in high-incidence settings

Line-probe Hybridization Assays

• Based on use of PCR amplification • Ffg by reverse hybridization • Hybridization assays designed as DNA-strips :

simultaneously detect MTB complex infx & anti-TB drug resistance

• Two commercial line-probe assays currently available • INNO-LiPA Rif TB assay can detect MTB complex & RIF

resistance • 2005 systematic review : sensitivities & specificities

ranging from 82 to 100 & 92 to 100% • Not able to detect additional anti-TB drug resistance

• Improved assay, GenoType® MTBDRplus • Common mutations in rpoB & katG & inhA genes • Recent meta-analysis : sensitivity & specificity for RIF

resistance 98.1 & 98.7% • Slightly lower for detecting INH resistance (84.3 &

99.5%) • Average time to analysis : 2 days - directly on sputum

+ samples • GenoType® MTBDRsl assay resistance to

fluoroquinolones, injectable drugs (capreomycin/amikacin/kanamycin) & EMB

• Targets gyrA, rrs & embB genes • Performs poorly for EMB resistance • Recent version 2

Hybridization assay disadvantages

• Focus on most prominent SNPs • Require well-trained staff & specialized laboratory infrastructure

• Technique has an open-tube format • Increased rate of false + results • Inapt tx of pxs

Molecular Beacons• Xpert MTB/RIF assay : can detect MTB

complex & associated RIF resistance : hemi-nested PCR

• 4.5 bacilli or 131 CFU/ml (in clinical specimens) per reaction & a TAT less than 2 h

• Disposable plastic cartridges • Fully automated • One manual step

REVIEW

• Multi-centre study: 98.2% sensitivity in smear+ clinical samples & 72.5% in smear- cases

• Reduced sensitivity in smear- cases improved to 90% when assay repeated 3x

• 99.1% sensitivity & 100% specificity compared with culture for detection of mutations in rpoB

• Performed in closed tube system • Disadvantage : Cost

PCR-based drug resistance dx tests hold a number of disadvantages

•Cost ,staff, contamination •Quality of specimen •Negative result should be interpreted with caution

•In Case of DST - not all the relevant resistance-causing mutations have been discovered

•Although specificity of NAATs remains high, the variable sensitivities should be addressed

•Drastic reduction in TAT - vast improvement over phenotypic-based assays

•NAATs able to detect nonviable bacteria as well

Loop-mediated Isothermal Amplification

• LAMP method : enhance or replace microscopy

• Auto-cycling strand displacement DNA amplification • Uniform temp by enzyme Bst polymerase • Requires only standard lab water bath or heating

block • Resulting stem-looped amplicons visually detected

by calcein • Increased specificity : multiple oligonucleotide

primers targeting same region • Enhanced DNA amplification : up to 50 x more than

standard PCR

• Numerous LAMP-based assays -detect MTB • Targets areas specific to mycobacteria • Including gyrB, rrs & rimM (encoding 16S rRNA-

processing protein) genes • Rapid (<90 min) • Highly specific , sensitive & requires minimal

infrastructure & lab skill • Only been developed to detect MTB • Currently no indication of drug susceptibility use

Transrenal DNA• Short fragments of mycobacterial DNA in

alternative specimens : urine • Urine : noninvasive & useful in dx of pxs

who cannot produce sputum • DNA arise from mycobacterial site &

transverse the renal barrier - excreted from the body in urine

• Transrenal DNA fragments detected by nested-PCR from soluble portion of urine

• Modified extraction & amplification

REVIEW

• Previous studies showed promising results in HIV-infected pxs

• Further evaluation necessary to confirm increased sensitivity

• Multicenter validation studies necessary to evaluate diagnostic accuracy in HIV-pxs & those with extrapulmonary dx

Immunological Methods

• Culture methods & genotypic-based assays---- high specificity : poor sensitivity

• Evident in paucibacilliary disease, smear- & extrapulmonary TB

• Host factors released by immune cells • Measure nonspecific mediators of

inflammation • T-cell-mediated immune response to MTB

antigens • Detects specific antibodies against these

antigens by serological tests

Latent M. tuberculosis Infection

• Low numbers of non replicating bacteria-dx only possible using immunological methods

• PPD - role in dx active dx • Limitations : poor sensitivity & specificity • Most widely used method to identify TB infx

without active dx • Size of TST induration : active TB

IFN-γ-release assays (IGRAs)

•Secretory antigenic target-6 & culture filtrate protein (CFP)-10

• IGRAS : blood tests recently developed as alternatives to TST

• Aid in the dx of latent TB infection

• Mainly used to dx LTBI in high-income countries, but increasingly being used to dx active TB in low-income & middle-income countries

• Two IGRAs in current use: QuantiFERON® -TB Gold In-Tube (QFT-GIT)

• T-SPOT®.TB (TSPOT)

REVIEW• Several published studies demonstrating

performance of IGRAs (Quantiferon TB Gold tests and T-SPOT.TB) over TST in dx of MTB

• Lack of reference standard test for LTBI • Problems • Constant scores (based on the gradient of

exposure to a TB index case)

• True accuracies - longitudinal studies • 2 studies - low TB incidence settings, non diseased

individuals initially evaluated with QFT assay ffg-up for 19 months & 2 years

• Individuals that progressed to active TB were initially QFT +

• High burden settings, useful in subject groups where MTB infection is difficult to dx

• TST performed better than IGRAs in some studies

Utility of Commercial IGRAs

• Remains questionable • High sensitivities • Lower specificities : inability to discriminate LTBI &

active • May be useful in dx of some forms of active TB, if

adapted • Direct measurement of IFN-γ in unmanipulated

pleural fluid using QFT - ELISA has shown to dx PTB with accuracy reaching 100% in studies

• ? clinically useful • Certain antigens have inadequate specificity

REVIEW

• Meta analysis • 27 studies (TSPOT 10 studies; QFT-GIT 17 studies)

involving 590 HIV-uninfected & 844 HIV-infected individuals

• With HIV-infection, pooled sensitivity estimates were modest: TSPOT 76% ; QFT-GIT 60%

• Without HIV-infection, pooled sensitivity estimates were higher: TSPOT 88% (4 studies); QFT-GIT 84% (9 studies)

• Specificity of both IGRAs was low (< 65%)

WHO POLICY• No role for IGRAs in dx of PTB in adults especially in high HIV

settings

• Policy implications: poor performance of current commercial IGRAs in low- & middle-income countries (typically high-TB &/or high HIV settings)

• Adverse impact of mis-dx & wasted resources on patients &health services when these tests are used for dx of active TB

• Systematic review: Metcalfe JZ et al. Interferon-gamma release assays for active pulmonary TB dx, J Infect Dis 2011

Role of Host Markers Other Than IFN-γ

• Host factors alone / combination with IFN-γ • IFN-γ inducible protein (IP)-10,IL-2 &

monocyte chemotactic protein-3 : bio markers • Remains questionable • Measurement of multiple biomarkers :

epidermal growth factor, macrophage inflammatory protein (MIP)-1β & IL-1α in TB-specific antigen-stimulated supernatants

• Limited reports

• The dx of smear- & extrapulmonary TB :CHALLENGING

• Biopsy ex : most sensitive method for dx of extrapulmonary TB

• Host markers in serum, urine or exhaled metabolites

• Easy-to-use dipstick-like test • Pleural fluid adenosine deaminase & IFN-γ most

evaluated markers for diagnosis of TB • High plasma levels of multiple inflammatory

markers found in pleural TB patients & levels of IP-10 & MIP-1α could - PTB vs EFFUSION

• Soluble ICAM-1 & neopterin

SEROLOGICAL TESTS• Crude cell preparations of MTB or M. bovis BCG • Highly purified native or recombinant antigens • 38 kDa antigen, antigen 60, lipoarabinomannan, members of the antigen 85 complex, MPT32 and MPT51

• Accuracies enhanced : use of combinations of antigen & multiple classes of antibodies

• Unavailability of useful commercial tests : may not be practical

• A number of antigens, including heat shock protein 65 shown to have some dx potential

• In comparison with microscopy, serological tests offer advantages of rapid results & simple technique

• In children & extrapulmonary dx : sputum difficult to obtain

• Despite the evidence, commercial serological tests for TB are in widespread use in high TB burden countries.

REVIEW 1

• 50% of studies performed in low & middle-income countries

• For PTB, 67 studies involving 5147 participants were included

• For all tests, estimates were variable for sensitivity (0% to 100%) & specificity (31% to 100%)

• For anda-TB IgG, the only serological test with enough studies for meta-analysis, pooled sensitivity was 76% in smear + (7 studies) & 59% in smear-(4 studies)pxs

WHO POLICY• Currently available commercial serological tests for TB are inaccurate

and inconsistent.

• Policy implications: In 2010 : negative recommendation against use of currently available serological tests

• Importance of continued research on serological & point-of-care tests

• Systematic review: Steingart KR et al. Commercial serological tests for the diagnosis of active pulmonary and extrapulmonary tuberculosis: An updated systematic review and meta-analysis. PLoS Medicine 2011

• Need to identify individual host markers or combinations of host markers

• Point of care tests• Enable accurate dx of active TB in

certain pxs • Diagnostic algorithm • Clinical & demographic characteristics

should be included

Expert Commentary & Five-year View In resource-limited settings

• sensitive • specific • Inexpensive • Point-of-care • Not require advanced training • Uses equipment that is easily transportable • Does not require mainline electricity

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