Diagnosis of TB: what is new from the laboratory

Daniela Maria Cirillo

San Raffaele Scientific Institute

Milano, Italy

Topics

• Introduction: role of the laboratory in TB diagnosis• Integrated laboratory network • Biosafety guidelines• Quality Assurance in the diagnosis of tuberculosis• Laboratory diagnosis of tuberculosis • Diagnostic algorithms• Conclusion

Introduction

• Care of TB patients starts with a QA diagnosis

• A robust network of Tb laboratories is required:– Adaquate biosafety– Modern diagnostics– SOPs– QAs

• Integrated laboratory network

Integrated laboratory network

An integrated laboratory network is:

able to provide all primary diagnostic services without requiring patients to go to different laboratory facilities for specific tests

ONE STOP SHOP

The past

• Weakest component of health systems for:– slow policy change and constant

underfunding – Inappropriate technology transfer– Inadequate infrastructure and biosafety– Staff skills and number– Inadequate lab strengthening plans– Insufficient technical assistance

The present

• Joint effort to improve and expand TB lab capacity integrated into a global health system strengthening

http://www.stoptb.org/wg/gli

• Research on new diagnostics tools accelerated and diagnostic pipeline rapidly growing ( expected point of care by 2015)

Quality Assurance Programme

System designed to continuously improve the reliability and efficiency of laboratory services

Three main components:

• Quality control (QC - IQA)

• External Quality Assurance (EQA)

» On site evaluation and review

» Proficiency Testing / Panel testing (PT)

» Blind rechecking

• Quality improvement (QI)

EQA Benefits• Allows comparison of performance and results

• Serves as an early warning-system for problems– Identifies systematic problems

• Provides objective evidence of laboratory quality

• Serves as an indicator of where to direct improvement efforts

• Identifies training needs

Laboratory biosafety

• Mtb is a class 3 risk pathogen• All biosafety strategies ( minimum requirements) should

be based on procedures risk assessment• Based on:

– Bacillary load of samples and workload– Viability of bacilli– Aeroosol generation– Typology of the patients– Fitness of the staff

MICROSCOPY

Rapid test

Inexpensive

Specificity: >95%

Sensitivity: 25-65% (90 % of infectious cases)

Positive Predictive Value for TB depends on epidemiological situation

1st AFB smear 80-82 %

2nd AFB smear 10-14 %

3rd AFB smear 5-8 %

Fluorescence Ziehl-Neelsen staining

Does not allow species identification

Not applicable to all samples

Excellent diagnostic test for TB control in high-incidence settings

LED Fluorescence Microscopy

Advantages:

• increase in performance

• increase in lamp lifetime

• reduces initial, operating and maintenance costs (adaptable to existing microscopes)

• No need for dark room

• Batteries operated

New Policy and Smear microscopy definition of a TB case

• New definition in 2007*:

“person with al least one smear-positive sample (1 AFB is sufficient) out of a total of two examined”

• 2 samples regardless the collection time*The definition/policy can be applied to countries performing microscopy under

satisfactory quality assurance programmes

Same day approach

same-day-diagnosis approach (microscopy of two consecutive spot-spot sputum specimens) is equivalent, in terms of diagnostic accuracy, to conventional case-finding strategies by microscopy

WHO recommends that:

Countries that have successfully implemented the two- specimen case-finding strategy consider a switch to the same-day-diagnosis approach, especially in settings where patients are likely to default from the diagnostic process

Countries that are still using the three-specimen case-finding strategy consider a gradual change to the same-day-diagnosis approach.

Changes to a same-day-diagnosis strategy be preceded by a detailed situation assessment

WHO recommendations on sputum smear microscopy (2010)

• ZN light microscopy performed on UNCONCENTRATED sputum is suitable for all laboratory service levels

• Concentration of sputum is NOT recommended in programmatic settings

• Fluorescence microscopy is recommended for increased sensitivity (add 10%)

• LED microscopy is recommended over conventional fluorescence

TB Culture*

Advantages• Definitive diagnosis of TB• Increases case finding of

30-50%• Early detection of cases• Provide strains for DST

and epidemiological studies

Disadvantages• Complex and expensive

compared to microscopy• Requires complex

handling of specimens• Skilled technicians• Appropriate infrastructure

and biosafety levels

LIMITATIONS: need for decontamination and identification

*coverage 500.000/1000000

Culture: solid/liquid

solid• Low cost for reagents, not

automated• Culture level infrastructure• Low contamination rate• Long time to positivity• Colony morphology• ID required

liquid• Complex and expensive can

be automated (MGIT)• Highest infrastructure and

biosafety levels• Case finding increased 10%

over solid• Diagnostic delay reduced to

days• ID required

Strip speciation tests needed for fast ID of TbcomplexMolecular test for speciation of most common mycobacteria

TB Culture: solid/liquid

• solid • liquid

Standardization of procedures still difficult to set up

The correct performance of DST requires the understanding of several steps such as:

Drug Susceptibility Testing (DST): Drug Susceptibility Testing (DST): the technical challenge still persiststhe technical challenge still persists

•Origin of resistance and interpretative criteria•Dosage and stability of the incorporated compounds•Anti-mycobacterial activity of the incorporate drug•Interpretation of results and data reporting

DST• Definitive diagnosis of DRTB

DST (2)

• Indirect methods suitable for regional/ national laboratories

• Adequate lab infrastructure and biosafety• Accuracy varies with the drugs tested:

– R, H most accurate– Second line :

• automated liquid DST are the gold standard• Not recommended in the absence of QA capacity (200 high

risk specimen per year)

• Formal link with Supranational Reference laboratories requested

Second line DST

• Recommended in appropriate structures for:– Aminoglycosides, Fluoroquinolones,

Polypeptides

• NOT recommended on routine for ethionamide, prothionamide, cycloserine,clofazimine, amox-clav, clarithromycin, linezolid)

WHO laboratory policies for culture and DST

• Automated liquid culture (2007) Use of liquid culture in the contest of comprehensive plan for strengthening lab capacity starting with national/central level

• Rapid speciation in combination with culture Second line DST (2008) to be conducted for selected drugs a central level

Available at http://www.who.int/tb.dots/laboratory/policy/en/print.html

Novel technologies for rapid screening of anti-TB drug

resistance have become a priority in Tuberculosis research

The effective treatment of MDR-TB is a life-saving intervention

Early diagnosis of both TB and DR-TB are the key for an effective Tb control

WHO policy on new diagnostics is evidence-driven

• Following new investments in research new diagnostics are finally moving

• Policy formulation needs to be an ongoing process at global and country level

• WHO asses policies by a systematic, structured process

Drug-Resistant TB: Definitions

• Mono-resistant: resistance to a single drug • Poly-resistant: resistance to more than one

drug, but not to the combination of isoniazid and rifampicin

• Multidrug-resistant (MDR): resistance to at least isoniazid and rifampicin

• Extensively drug-resistant (XDR): MDR plus resistance to fluoroquinolones and at least 1 of the 3 injectable drugs (amikacin, kanamycin, capreomycin)

Drug-Resistant TB: Definitions

MONORESISTANT

Drug-Resistant TB: Definitions

POLIRESISTANT

Drug-Resistant TB: Definitions

MULTI DRUG RESISTANT

Drug-Resistant TB: Definitions

???MULTI DRUG RESISTANT

Drug-Resistant TB: Definitions

EXTENSIVELY DRUG

RESISTANT

Impact of Resistance on Outcome

Resistance pattern New Cases (%) Retreatment (%)

Pan-susceptible 4 10

Any Resistance 5 21

MDR 30 45

INH (not MDR) 6 23

RIF (not MDR) 13 29

Other 4 15

% of cases with failure or death, standard 4-drug regimen

Espinal MA, et al. JAMA. 2000;283(19):2537-45

Mechanisms of resistance

• Production of enzymes that modify or destroy the antibiotic molecule

• Alteration of the target binding site

• Active escretion of the molecule (efflux pump)

• Membrane permeability reduction

INH = 1 in 106

RIF = 1 in 108

EMB = 1 in 106

Str = 1 in 106

I + R = 1 in 1014

Frequency of Resistance Mutations

Genes involved in drug-resistance for major Genes involved in drug-resistance for major anti-tubercular drugsanti-tubercular drugs

PAGE | 36

RIF resistance as surrogate marker for MDR TB

• RIF resistance as a mono resistance not very frequent (5-15% of them)

• 80-95% of RIF resistant strains are also resistant to INH• Key-drug in the anti-TB regimen• Low rate of spontaneous mutations• Mutations affect an hot-spot region in the rpoB gene

Khue et al. A 10-year prospective surveillance of Mycobacterium tuberculosis drug resistance in France 1995–2004. Eur Respiratory J. 2007

Ideal candidate for the molecular detection of resistance to the drugIdeal candidate for the molecular

detection of resistance to the drug

Commercial Line Probe AssaysCommercial Line Probe Assays

Hain LifescienceHain Lifescience

InnogeneticsInnogenetics

INNO-LiPA-INNO-LiPA-Rif.TBRif.TB

GenoType® MTBDR

INNO-LiPA Rif.TB

Company Hain Lifescience

Innogenetics

M. tuberculosis detection Yes Yes

Detection of RMP Resistance in M. tb Complex

Yes Yes

Detection INH Resistance in M. tb Complex

Yes No

Strip Assay Yes Yes

PCR based Yes Yes

From liquid or solid culture Yes Yes

Direct assay Yes Yes (modified version)

TBCDetection: 23S-rRNA/16S-rRNA Yes Yes

RMP-Resistance: rpoB gene Yes Yes

INH-Resistance: katG gene/inhA gene Yes No

Universalcontrol Yes No

rpoB unicontrol Yes No

kat G/inHA unicontrol Yes No

Comparison GenoType® MTBDRplus and INNO-LiPA Rif.TB

New generation of LiPA performs better in both Sm+ and Sm- samples

Miotto al. JCM 2008

Molecular line probe assays for rapid screening of patients at risk of MDR-TB

Policy statement by WHO and Partners

June 27, 2008

Endorsement of the two commercial line probe assays for rifampicin resistance detection:Tests are CE marked and meet predefined performance targets in controlled evaluation studiesBoth tests are highly sensitive and specific for rifampicin resistance detection from TB strains

Smear positive Smear negative

Rif/ Rif INH LiPA

MTB detected, INH(*) and RMP susceptible

MTB detected, INH and/or RMP resistant (MDR-TB)

•MTBC, INH(*) and RMP mutation non detected NON MDR•No culture

Start standard 1st line treatment

Culture (liquid/ solid)DST ( first and second line)

Report as negative OR if TB is highly suspected (HIV+,child..)

perform (liquid) culture

Culture AFB positive, TB morphology

Unsuccessful amplification

Perform (liquid) culture

Culture AFB positive

ID

Sputum samples

Monitoring sputum conversion

Standard MDR treatment

Individualized treatment

Treatment with 2nd-3rdDrugs available/isolation

monitoring

Possible automation on LiPA

LiPAs require:Level II biosafety areasSkilled laboratory staffAmplicon Contamination control

•simple 1-step external sample prep. •time-to-result < 2 h •throughput: >16 tests / day / module •no need for biosafety cabinet •integrated controls •true random access •specific for MTB •sensitivity similar to culture •detection of rif-resistance via rpoB gene •test cartridges for GeneXpert System •several GeneXpert modules can be combined in 1 workstation •~1 day technician training required

XPERT TB-MDR

Simple Sample Processing – Direct Sputum

1. Add 2:1 Sample Buffer to sample

2. Shake then stand 10 minutes

3. Shake then stand further 5 minutes

4. Transfer 2mlto cartridge

Begin Test…

Moving to “district” :Dharamsala, Delek Hospital

Sensitivity (cfu/ml) of pulmonary TB diagnostics

Solidculture

Advantages

• Simple to perform• Minimal training• Virtually cross contamination free• Minimal biosafety requirement• Higher sensitivity in paucibacillary samples

(HIV+)

Proportion of TB cases detected

Time to detection

Time to RFP resistance detection

Potential limits of Xpert MTB/RIF technology

• Unknown the performance at a district level• If RFP resistance is diagnosed at a low level MDR prevalence

environment, the assay needs to be confirmed • Need to perform a culture for DST to evaluate other drug resistance• Need to perform a culture for monitoring issue (culture conversion)• It requires uninterrupted and stable electronic power supplies and

yearly calibration • Size for storage issues

New WHO recommendations

WHO RECOMMENDATION, December 8th, 2010

New WHO recommendations

WHO RECOMMENDATION, December 8th, 2010

New WHO recommendations

WHO RECOMMENDATION, December 8th, 2010

GenoType® MTBDRplus

XpertTB-MDRplus

Company Hain Lifescience

Cepheid

M. tuberculosis detection Yes Yes

Detection of RMP Resistance in M. tb Complex

Yes Yes

Detection INH Resistance in M. tb Complex

Yes No

Fully automated /training No/Yes Yes/No

DNA tech PCR Mol Beacon

From liquid or solid culture Yes NA

Direct assay Yes Yes

Level of biosafety II microscopy

Time to results Same day 2h

Cost per test Low/Mod Mod/High

Universal control Yes Yes

Extraction control No Yes

Cost of MaintenanceContamination control

LowNo

HighYes

Comparison GenoType® MTBDRplus and XpertTB/MDR

Intermediate Reference labsPatients testing from Sm/ C+ (Rif)Fast tool Surveillance purposes

Potential to District level as fast patients diagnostic tool, needs evaluation at district level

Smear positive Smear negative

Rif/ Rif INH LiPA

MTB detected, INH(*) and RMP susceptible

MTB detected, INH and/or RMP resistant (MDR-TB)

•MTBC, INH(*) and RMP mutation non detected NON MDR•No culture

Start standard 1st line treatment

Culture (liquid/ solid)DST ( first and second line)

Report as negative OR if TB is highly suspected (HIV+,child..)

perform (liquid) culture

Culture AFB positive, TB morphology

Unsuccessful amplification

Perform (liquid) culture

Culture AFB positive

ID

Sputum samples

Monitoring sputum conversion

Standard MDR treatment

Individualized treatment

Treatment with 2nd-3rdDrugs available/isolation

monitoring

Xpert-TB/MDR-TB

Contact tracing?

Genes involved in drug-resistance for major Genes involved in drug-resistance for major anti-tubercular drugsanti-tubercular drugs

40-80%

XDR molecular diagnosis: GenoType MTBDRsl (Hain Lifescience)

Technologies and laboratory appropriateness

• Introducing new technology requires addressing of core elements:– Infrustructure, biosafety measures and maintenance– Equipment validation and maintenance– Specimen transport and referral mechanisms– Management of laboratory commodities and supplies– Laboratory information data and management system– Laboratory quality management– Strategies for HR development and retentionGLI road map at

:http://www.who.int/tb/dots/laboratory/policy/en

“Point of care assay”

Closer if• Large scale innovation and delivery

will be fully supported by:–Scientists and industry–Large operational research trials–Large view policy makers and regulators–Retooling of existing resources