1. Second Line Drugs Susceptibility Testing: Study Progress Report Alex Sloutsky Massachusetts State TB Laboratory Paris, 2005

2. B ACKGROUND: Clinical significance of DST It is common among clinicians to assume that if culture is resistant to a particular drug in vitro, this drug will be ineffective in vivo. In reality, prognostic value of pretreatment DST can vary greatly depending on patient-specific factors: - proportion of resistant bacteria - whether resistance is acquired or primary Clinical significance of susceptibility testing becomes an issue due to spread of MDR TB.

3. Resiissstaannsensitivity DST reports list drugs as either “sensitive” or “resistant” which indicates that there are two widely different phenotypes. In reality, resistant strains exhibit a continuum of phenotypes, some of which may be very similar to sensitive, wild-type strains. The task of the laboratory is to classify the level of resistance of a strain of M. tuberculosis in relation to the established “resistance criteria” to a specific drug. These “breakpoints” determine clinically significant level of resistance to a drug, i.e. high enough to affect the efficacy of treatment if the patient is treated with that drug

4. Study Design: MSLI Lab Well-defined representative samples of clinical isolates of M. tuberculosis: –PR strains have been obtained from patients who failed treatment with the regimens containing the corresponding drug; –Probably susceptible (PS) strains: from patients who have never taken anti-TB drugs (unless infected with drug resistant organisms). Source of strains: KIT (Korea), Philippines (TDF), Latvia, Hong Kong, Boston (PIH collection). Total planned:250 strains. MIC to 6 drugs are to be determined: ETH, PAS, KAN, CAP, CYC, OFL by two methods: Agar Plate Proportions and BACTEC. 8 concentrations of each drug used to determine the MIC breakpoint.

5. WORKLOAD AND WORKFLOW: Agar Plate Proportions Method Determination of Critical Concentrations on agar medium Weekly workload Time to complete Preparation of the four-quadrant plates with drug dilutions: 3 plates per each strain per one drug (4,500 plates total) 108 plates4 hours Preparation of 2nd line drug dilutions (6 drugs, 8 concentrations each): 48 dilutions1 hour Growing cultures, inoculum preparation6 strains2 hours Inoculation of the 7H10 agar plates including labeling 108 plates2 hours Reading plates and recording results108 plates5 hours Total time allotted per week is 14 hours

6. WORKLOAD AND WORKFLOW: BACTEC Determination of Critical Concentrations in BACTEC Weekly workload Time to complete Preparation of BACTEC vials with serial drug dilutions including labeling Total number required is: 250 strains x 6 drugs x 8 concentrations plus controls = 13,500 vials; 288 vials4 hours Inoculum preparation, inoculate drug- containing BACTEC vials plus controls; 294 vials4 hours Five daily BACTEC runs, 1.5 h each, using 2 instruments; includes Sat; (6 hours per day) 294 vials x 6 days =1,764 36 hours Recording BACTEC results2 hours Total time allotted per week for BACTEC is 46 hours

7. BUDGET

8. PROJECTED DURATION AND CURRENT STATUS OF THE PROJECT Total time per week for 6 strains tested by both methods is 60 hours. It was anticipated that it would take 250/6 = 42 weeks to completion (with no repeat testing and no data analysis). Up to date: 272 cultures tested by APP method (< 10 to be repeated) and 40 tested by BACTEC. ~230 cultures to go will take ~40 weeks or 10 months Personnel hired and trained Supplies purchased Forms for recording results created

9. DATABASE

10. Preliminary Data Analysis