Second Line Drugs Susceptibility Testing: Study Progress Report

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

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BACKGROUND 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.

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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

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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 planned250 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.

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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 plates 4 hours
Preparation of 2nd line drug dilutions (6 drugs, 8 concentrations each) 48 dilutions 1 hour
Growing cultures, inoculum preparation 6 strains 2 hours
Inoculation of the 7H10 agar plates including labeling 108 plates 2 hours
Reading plates and recording results 108 plates 5 hours
Total time allotted per week is 14 hours
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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 vials 4 hours
Inoculum preparation, inoculate drug-containing BACTEC vials plus controls 294 vials 4 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 results 2 hours
Total time allotted per week for BACTEC is 46
hours
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BUDGET
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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).
Personnel hired and trained Supplies
purchased Forms for recording results created
Up to date 272 cultures tested by APP method (lt
10 to be repeated) and 40 tested by BACTEC. 230
cultures to go will take 40 weeks or 10 months
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DATABASE
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Preliminary Data Analysis