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

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

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

5. 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.

6. 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

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

8. 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.

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

10. TAT After Implementation of New Methods ProcedureRequired TAT (days) Real TAT (days) Improved TAT Smear111 Detection of growth 1420-605-30 Identification2135-907-35 DST2850-9014-42 30 (dir)

11. Factors That Influence Implementation of New Methods Reduction of the TAT and its impact on patient management. Costs. –Upfront cost for equipment; –Long-term cost increase for supplies and reagents; Complexity. –Does your laboratory have the basics? –Are your staff trained enough? How does the new method fit into your lab work flow and personnel schedules.

12. 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

13. 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

14. Other Important Variables Which Can Reduce TAT but are Easy to Forget Post-analytic part (3) Pre-analytic part (1) Analytic part (2) Test ordering Specimen collection Specimen transport Specimen receiving & processing Testing Results review and follow- up Interpretation Reporting Data management Specimens storage Overall TAT = TAT 1 + TAT 2 + TAT 3 Specimens Flow Path

15. QUALITY CONTROL (QC)

16. 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

17. QC/QA: BRIDGING THE GAP BETWEEN NTP AND LAB SERVICES NTP: CLINICAL COMMUNITY, TB CONTROLLERS, EPIDEMIOLOGIS TS 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 ARE QUALITY CONTROL

18. OPERATIONAL RESEARCH IMPROVING ROUTINE DIAGNOSTICS

19. 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. MTD posneg pos3515 neg 248 culture False-neg False-posTrue-neg True-pos

20. Improvement of MTD test performance MTD posneg pos3715 Neg 263 culture False-neg False-posTrue-neg True-pos NEW SENSITIVITY = 100% NPV = 100%

21. 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

22. 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