CLINICAL DIAGNOSTIC TB LABORATORY

1. CLINICAL DIAGNOSTIC TB LABORATORY Alexander Sloutsky, Director University of Massachusetts Supranational Reference TB Laboratory Boston, MA

2. ASSESSMENT OF THE TB LABORATORY SERVICES • Currently used methods and new methods implementation • Turn-around time (TAT) and CDC guidelines • Quality Control/Quality Assurance

3. Functions of Large TB Diagnostic Lab Operational research International projects QA/QC Diagnostics

4. 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.

5. 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

6. CURRENTLY USED ALGORITHM FOR DETECTION AND ID OF MYCOBACTERIA NaOH-NALC: liquefaction/ decontamination Sputumor other sample Concentration by centrifugation 1 day 1 day Set-up for growth Smear (acid-fast microscopy) Sediment Solid media colonies 3-6 weeks Liquid media 7-14 days Culture 1 day Growth Isolates MTD (NAA amplification test) 4-6 weeks 1 day 1 day ID Conventional biochemical tests Nucleic acid probes HPLC of mycolic acids Species

7. 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.

8. 20 = days to complete AFB Smear Detection Detection Improved TAT for TB Lab Workflow Direct DST susceptibility testing 4-30 1 28 Specimen processed Culture liquid DST 14-42 Culture solid 7-35 1 BACTEC 10-12 ID for TB identification

9. TAT After Implementation of New Methods

10. 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

11. 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

12. Pre-analytic part (1) Analytic part (2) Post-analytic part (3) Other Important Variables Which Can Reduce TAT but are Easy to Forget Specimens Flow Path • 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

13. QUALITY CONTROL (QC)

14. Quality Control (QC) • Process of effective and systematic control for all laboratory activities which helps to eliminate restrictions 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

15. QUALITY CONTROL QC/QA: BRIDGING THE GAP BETWEEN NTP AND LAB SERVICES NEED SERVICE PROVIDE SERVICE NTP: CLINICAL COMMUNITY, TB CONTROLLERS, EPIDEMIOLOGISTS TB LABORATORIES OF DIFFERENT LEVELS UNITED BY EXTENSIVE SYSTEM OF QC/QA QC: HELPS TO UNDERSTAND WHAT CAN BE EXPECTED FROM THE LABORATORY AND WHAT CAN NOT QC: HELPS TO UNDERSTAND WHAT THE EXPECTATIONS ARE

16. OPERATIONAL RESEARCH IMPROVING ROUTINE DIAGNOSTICS

17. Versus culture: sensitivity - 70%; specificity >95% PPV ~ 80%, NPV ~ 86%; False-positives: cross-contamination (affect specificity) False-negatives: reaction inhibitors (affect sensitivity) MTD test performance culture False-neg True-pos False-pos True-neg • Clinically, sensitivity 70% means that 30% of the patients are undiagnosed for several weeks.

18. Improvement of MTD test performance culture False-neg True-pos False-pos True-neg NEW SENSITIVITY = 100% NPV = 100%

19. Impact of improved MTD test on TB control in patients with high clinical suspicion of TB NO NO NO Modified from Michael Iseman’s book “A Clinician’s Guide toTuberculosis”

20. 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