Second Line Drugs Susceptibility Testing: Study Progress Report

Alex SloutskyMassachusetts State TB Laboratory

Paris, 2005

BBACKGROUND: Clinical significance of ACKGROUND: Clinical significance of DSTDST

• 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.

ResiissstaannsensitivityResiissstaannsensitivity• 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

Study Design: MSLI LabStudy 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.

WORKLOAD AND WORKFLOW: Agar WORKLOAD AND WORKFLOW: Agar Plate Proportions MethodPlate 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

WORKLOAD AND WORKFLOW: WORKLOAD AND WORKFLOW: BACTECBACTEC

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

BUDGETBUDGETA. Personnel Time allotted Salary TOTAL

Contractor 111 hours per week @ $35.50/hour

$20,306

Contractor 212 hours per week @ $22.50/hour

$14,040

NEW, Bacteriologist I 37.5 h/week @ $19.50/hour

$38,025

TOTAL for personnel $72,371 $72,371

B. Laboratory Supplies Cost

Bactec Supplies $3,000

Bactec 12B Medium (for 13,750* vials @ $2.27 each)

$31,212

7H10 agar plates for CC determination (4,500* plates @ $4 ea.)

$18,000

General supplies $3,000

Total for supplies $55,212

Travel & administrative $4,000 $4,000

$131,583

PROJECTED DURATION AND CURRENT PROJECTED DURATION AND CURRENT STATUS OF THE PROJECTSTATUS 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

DATABASEDATABASE

Shortcut to frm_Menu in WHO_DATA.MAF

Preliminary Preliminary Data AnalysisData AnalysisCONC CYC CAP ETH KAN OFX PAS

1 99.3% 97.1% 57.7% 83.8% 88.6% 54.0%2 90.8% 75.4% 55.9% 61.8% 39.0% 44.9%3 29.8% 41.5% 49.3% 46.7% 24.6% 38.2%4 1.1% 29.8% 43.0% 39.0% 22.4% 28.3%5 0.4% 25.0% 39.0% 32.7% 20.6% 22.1%6 0.4% 19.1% 37.1% 29.8% 15.8% 21.0%7 0.0% 15.1% 31.3% 26.8% 12.5% 19.1%8 0.0% 5.9% 28.3% 26.1% 9.6% 18.0%

12

34

56

78

CYC

ETH

OFX0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

CYC

CAP

ETH

KAN

OFX

PAS