CLINICAL DIAGNOSTIC TB LABORATORY

Alexander Sloutsky, Director

University of Massachusetts Supranational Reference TB Laboratory

Boston, MA

ASSESSMENT OF THE TB LABORATORY SERVICES

• Currently used methods and new methods implementation

• Turn-around time (TAT) and CDC guidelines

• Quality Control/Quality Assurance

Functions of Large TB Diagnostic Lab

Ope

ratio

nal

rese

arch

International

projects

Diagnostics

QA/QC

USERS: Who Needs TB Laboratory Services?

• Clinical Community (hospitals, individual doctors);• Public Health practitioners;• Social Institutions (penitentiary system, shelters,

nursing homes and assisted living facilities, refugee health, and other w/congregated living);

• Research and Development teams from pharmaceutical companies, other research institutions;

• Non-profit organizations launching international TB-related projects in low-income countries.

METHODS

• Detection of acid-fast bacilli (AFB) primarily in respiratory specimens; also in other body fluids or tissues.

• Smear preparation and microscopy• Processing specimens• Detection of AFB growth in liquid and solid cultures• Molecular detection (PCR, MTD and other methods)

• Identification to species • Conventional (biochemical) methods• Molecular probes • HPLC• DNA-sequencing of highly polymorphic regions, amplified by PCR

• Drug Susceptibility testing (DST)• Conventional (Agar Plate Proportion)• Rapid ( BACTEC) • Molecular methods

CURRENTLY USED ALGORITHM FOR DETECTION AND ID OF MYCOBACTERIA

Sputum or other sampleNaOH-NALC:liquefaction/

decontamination

Concentration by centrifugation

Smear (acid-fast microscopy)Sediment

Culture

Species

Conventional biochemical testsNucleic acid probes

HPLC of mycolic acids

ID

1 day

Solid media colonies3-6 weeks

Set-up for growth

1 day

Liquid media7-14 days

4-6 weeks1 day1 day

MTD (NAA amplification

test)

1 day

Isolates Growth

New Methods in TB Diagnostics

Q.: What drives people’s desire to develop and implement new methods for TB diagnostics?

A.: Reduction of the TAT and its impact on patient management.

Improved TAT for TB Lab Workflow

Specimenprocessed

AFBSmear

Culture solid

ID for TB

DST

1

14-42

20 = days to complete

7-35

Culture liquid

4-30

1

DirectDST

28

BACTEC 10-12

TAT After Implementation of New Methods

Procedure Required TAT (days)

Real TAT (days)

Improved TAT

Smear 1 1 1

Detection of growth

14 20-60 5-30

Identification 21 35-90 7-35

DST 28 50-90 14-42

30 (dir)

Rapid detection of DR TB: gains

• Urban populations with significant prevalence of MDR TB and HIV co-infection, where data on the resistance genotype may result in better patients management

• Congregate living facilities where a quick action has to be taken

• Culture is not available but the answer is very important– Exposure of a large group of cancer patients to a

TB case

Rapid detection of DR TB: pains

• All molecular techniques are based on NAA (PCR) which will gladly amplify any DNAs including contaminating ones…

• Each case falsely diagnosed with DR TB will receive unnecessary treatment with second line drugs

• Cost for molecular testing is high. Difficult to include into lab algorithm (batching vs. TAT)

• Except Rifampin, testing for R to all other TB drugs have complications

Other Important Variables Which Can Reduce TAT but are Easy to Forget

Post-analytic part (3)

Pre-analyticpart (1)

Analyticpart (2)

•Test ordering

•Specimen collection

•Specimen transport

•Specimen receiving & processing

Testing

Results review and follow-up Interpretation

Reporting

Data management

Specimens storage

Overall TAT = TAT1 + TAT2 + TAT3

Specimens Flow Path

QUALITY CONTROL (QC)

Quality Control (QC)

• Process of effective and systematic control for all laboratory activities which helps to eliminaterestrictions for quality testing

• Good QC program provides warranty for accuracy,reliability and comparability of laboratory results.

• Mechanism which helps to verify proficiency of TB diagnostic Services.

• QC is responsibility of all Lab staff and management

QC/QA: BRIDGING THE GAP BETWEEN NTP AND LAB SERVICES

NTP: CLINICAL COMMUNITY, TB CONTROLLERS,

EPIDEMIOLOGISTS

TB LABORATORIES OF DIFFERENT

LEVELS UNITED BY EXTENSIVE SYSTEM

OF QC/QA

NEED SERVICE PROVIDE SERVICE

QC: HELPS TO UNDERSTAND WHAT CAN BE

EXPECTED FROM THE

LABORATORY AND WHAT CAN

NOT

QC: HELPS TO UNDERSTAND

WHAT THE EXPECTATIONS

AREQUALITY CONTROL

OPERATIONAL RESEARCH IMPROVING ROUTINE DIAGNOSTICS

MTD test performance

• Versus culture: sensitivity - 70%; specificity >95%PPV ~ 80%, NPV ~ 86%;

– False-positives: cross-contamination (affect specificity)– False-negatives: reaction inhibitors (affect sensitivity)

• Clinically, sensitivity 70% means that 30% of the patients are undiagnosed for several weeks.

MTDpos neg

pos 35 15

neg 2 48

culture

False-neg

False-pos True-neg

True-pos

Improvement of MTD test performance

MTDpos neg

pos 37 15

Neg 2 63

culture

False-neg

False-pos True-neg

True-pos

NEW SENSITIVITY = 100%NPV = 100%

Without MTD

With MTD

Treatment yes yes

Isolation yes yes

Contact investigation

yes yes

Treatment yes ?

Isolation yes ?

Contact investigation

yes ?

Potential action

positive

negative

Smear-positiveMTD Results

Impact of improved MTD test on TB control in patients with high clinical suspicion of TB

Modified from Michael Iseman’s book “A Clinician’s Guide toTuberculosis”

NO

NO

NO

Examples of research projects in TB Lab• Evaluation, validation and implementation of

molecular diagnostics by broad spectrum PCR and a follow-up DNA-sequencing in:– liquid media, when there is not enough growth to use

Molecular Probes

– paraffin-embedded tissue specimens– Primary specimens Also, PCR and DNA-sequencing of specific mutation conferring

resistance to INH, ETH, Rif, PZA, FQs (in lieu of DST)

• Development of an original method for assessment of quality of sputum specimens.

• RFLP typing for detection of cross-contamination.• Development of new medium improving recovery of

Mycobacteria from sputum specimens.• Study of cross-resistance between aminoglycosides as well

as between INH and Ethionamide