Download WORKLOAD AND WORKFLOW: 6 strains per week

Second Line Drugs Susceptibility
Testing: Study Progress Report
Alex Sloutsky
Massachusetts State TB Laboratory
Paris, 2005
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.
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
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.
WORKLOAD AND WORKFLOW: Agar
Plate Proportions Method
Determination of Critical
Concentrations on agar medium
Weekly
workload
Preparation of the four-quadrant plates 108 plates
with drug dilutions: 3 plates per each
strain per one drug (4,500 plates total)
Preparation of 2nd line drug dilutions
(6 drugs, 8 concentrations each):
Time to
complete
4 hours
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:
BACTEC
Determination of Critical Concentrations
in BACTEC
Weekly
workload
Time to
complete
Preparation of BACTEC vials with serial
drug dilutions including labeling
288 vials
4 hours
Inoculum preparation, inoculate drugcontaining 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
Total number required is: 250 strains x
6 drugs x 8 concentrations plus controls
= 13,500 vials;
Recording BACTEC results
2 hours
Total time allotted per week for BACTEC is 46 hours
BUDGET
A. Personnel
Time allotted
Salary
Contractor 1
11 hours per week
@ $35.50/hour
$20,306
Contractor 2
12 hours per week
@ $22.50/hour
$14,040
NEW, Bacteriologist I
37.5 h/week @
$19.50/hour
$38,025
TOTAL for personnel
B. Laboratory Supplies
Bactec Supplies
Bactec 12B Medium (for
13,750* vials @ $2.27
each)
7H10 agar plates for CC
determination (4,500*
plates @ $4 ea.)
General supplies
$72,371
$72,371
Cost
$3,000
$31,212
$18,000
$3,000
Total for supplies
Travel & administrative
TOTAL
$4,000
$55,212
$4,000
$131,583
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 (< 10 to
be repeated) and 40 tested by BACTEC. ~230 cultures
to go will take ~40 weeks or 10 months
DATABASE
Preliminary Data Analysis
CONC
1
2
3
4
5
6
7
8
CYC
99.3%
90.8%
29.8%
1.1%
0.4%
0.4%
0.0%
0.0%
CAP
97.1%
75.4%
41.5%
29.8%
25.0%
19.1%
15.1%
5.9%
ETH
57.7%
55.9%
49.3%
43.0%
39.0%
37.1%
31.3%
28.3%
KAN
83.8%
61.8%
46.7%
39.0%
32.7%
29.8%
26.8%
26.1%
OFX
88.6%
39.0%
24.6%
22.4%
20.6%
15.8%
12.5%
9.6%
PAS
54.0%
44.9%
38.2%
28.3%
22.1%
21.0%
19.1%
18.0%
100.0%
80.0%
CYC
60.0%
CAP
40.0%
ETH
KAN
20.0%
OFX
0.0%
1
OFX
2
3
4
ETH
5
6
7
CYC
8
PAS