Transcript KMK_SLDDSTProposal []

What is needed for standardizing
second line drugs testing?
SLCS/ SRLN Meeting,
Institut Pasteur
Paris, October 23-24, 2005
TB Reference Laboratory,
Department of Health,
Hong Kong.
Indications for culture
(1998)
1. Diagnosis of cases with clinical and /or
radiological signs of TB that are repeatedly
smear negative
2. Failures and chronic cases
3. Drug resistance to establish the resistance
pattern either for survey or for the implementation
of DOTS-Plus
4. Diagnosis of extra-pulmonary TB
5. TB in children
6. As a gold standard in research and before
implementation of new diagnostic tools
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3
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Table 3. Summary of biosafety level requirements
BIOSAFETY LEVEL
Isolationa of laboratory
Room sealable for decontamination
Ventilation: — inward airflow
— controlled ventilating system
— HEPA-filtered air exhaust
Double-door entry
Airlock
Airlock with shower
Anteroom
Anteroom with shower
Effluent treatment
Autoclave: — on site
— in laboratory room
— double-ended
Biological safety cabinets
Personnel safety monitoring capabilityd
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No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
a Environmental and functional isolation from general traffic.
b Dependent on location of exhaust (see Chapter 4).
c Dependent on agent(s) used in the laboratory.
d For example, window, closed-circuit television, two-way communication.
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3
No
Yes
No
Yes
Desirable
Yes
Desirable
Yes
No
Yes/Nob
No
Yes
No
No
No
No
No
Yes
No
Yes/Noc
No
Yes/No c
Desirable
Yes
No
Desirable
No
Desirable
Desirable
Yes
No
Desirable
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Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
—
No
Yes
Yes
Yes
Yes
Yes
Yes
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WHO Biosafety Manual, 2004
So,
• In pursuing standardization of anti-TB
SLDSTs , do we need a BSL 3 facility as a
standard practice for labs doing DOTS plus
implementation??
Or, …..?
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What is the purpose of standardizing second line
anti-TB drugs testing?
__________________________________________
(1) Can clinical relevance of criteria of resistance be ensured?
(2) Can reliable laboratory test procedures be found?
(3) Can inter-laboratory comparison be facilitated?
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Organization and Planning
Prior to calibration of the DST testing methods:
1.
(i)
Careful selection of TB institutes or TB hospitals, that
will participate in collection of specimens and MTB
isolates from patients
(ii) with accurate history of disease and anti-TB treatment.
2.
Careful selection of laboratories with capability,
interest, and commitment, <and can communicate well
with clinicians and with each other>.
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3.
(i)
(ii)
(iii)
Representative sample of M. tuberculosis strains
Most important issue;
Well-documented;
From patients under treatment with regimens
containing the interested drug for 6 months or more
(PR), and those who have never taken any anti-TB
drugs before (PS).
(iv) Total sample size should be large enough, i.e. around
400 strains.
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4. Requirements for quality control
a.
b.
Internal quality control – media, H37RV, drug
concentrations
External quality control
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5. Level of resistance (or susceptibility)
investigated with all cultures by determining
MIC in the various media in most common use
for routine services, and the proportion of
resistance at the different levels of drug
concentrations on the various solid media.
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6. Different physicochemical test environments
influence in vitro results to a great extent,
Therefore, desirable to investigate factors that
influence test results most seriously in the method
and drug under consideration.
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7. Outcome is to develop and elaborate a
standardized laboratory test protocol using
available techniques that can be used under
program settings.
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1. Culture collection
Well-defined and representative samples of clinical
isolates of M. tuberculosis be used in calibrating
of DST method that is being used to determine
clinically relevant and technically feasible in vitro
resistance criteria of the second line drugs.
<? Standard/ Agreed format for nomenclature of
study strains for inter-laboratory comparison>
<Broader availability of strains>
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(1) Collection of probably resistant strains:
These strains should be collected with extreme care.
In general, PR strains are those isolated from patients who
apparently failed with regimens containing the
corresponding drug because of continuous
expectoration of live MTB bacilli despite taking the drug
for at least 6 months at the time of strain isolation from
the clinical specimens.
Drugs taken previously and not under current use for at
least 6 months will not be taken into account.
The level of resistance and other biological characteristics
of the strains may vary from region to region or from
setting to setting. However, these will not be taken into
account.
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(2) Collection of probably susceptible strains:
These strains are derived from patients who have
never taken anti-TB drugs so they are probably
susceptible to all anti-TB drugs unless patients
have been infected with drug resistant
organisms.
MTB strains with primary drug resistance or with
natural resistance will not be included for
calibration of test procedure to determine the
resistance criteria.
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(3) Subculture and storage of the strains:
MTB strains selected are subcultured onto
butt medium prepared in cryotubes (at
least 15 tubes per strain) and stored at
freezer or deep freezer (≤-50℃) until they
are to be tested.
Patient’s details and the name of drugs
taken for 6 months or more at the time of
isolation are recorded.
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2. Investigation on the in vitro criteria of resistance
Factors that may lead to incorrect laboratory
results are:
(1) failure in control of inoculum standardization,
(2) use of clinically irrelevant criteria of resistance,
and/or
(3) failure in stabilization or control of
physicochemical test environment.
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Failure in inoculum control is usually related to:
(1) variation of inoculum size,
(2) dispersion of bacillary clumps, and
(3) viability of bacilli in the inoculum.
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Use of improper resistance criteria, which have not
been carefully calibrated with clinical isolates of
PR and PS MTB, will lead to incorrect results,
incorrect interpretation, incorrect treatment
regimens, loss of credibility of program.
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Many of the second line drugs suffer from inherent
difficulties in obtaining reliable resistance criteria
because their MICs are close to peak blood levels so
that, in some patients, drug remains in subinhibitory
concentration in the lesions in which selective
multiplication of resistant mutants can take place.
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Metabolic activity of bacilli in the lesions and
antibacterial activity of drugs both directly
influence the selective growth of drug resistant
mutants.
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DST results are also greatly affected by physicochemical
properties of test environment:
(1) drug potency and dissolution,
(2) heat inactivation,
(3) protein binding,
(4) presence of antagonists,
(5) pH,
(6) humidity, temperature, and duration of storage of drug
containing media.
DST procedure, media should be carefully set up and
calibrated to obtain clinically relevant test results taking
into account these factors.
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OBJECTIVES
The project aims to :
1. Determine clinically relevant in vitro resistance criteria
of the reserved anti-tuberculosis drugs;
2. Facilitate obtaining reliable prevalence data of the
reserved anti-TB drug resistance;
3. Better assist clinicians in selecting the effective
treatment regimen (individualized or standardized) for
MDR-TB cases; and
4. Set up of laboratory test environment that can improve
reproducibility and reliability of the test results.
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Table 2. Anti-TB drugs and their concentrations to be tested
Drug concentrations (㎍/㎖) to be tested
Media
L-J
Medium
KM
CPM
PTH
CS
OFX
PAS
5
10
10
10
0.25
0.125
10
20
20
20
0.5
0.25
20
30
30
30
1
0.5
30
40
40
40
2
1
40
50
50
50
3
2
60
60
60
60
4
3
80
80
80
80
5
4
120
120
120
120
6
6
Stability of pH of egg-based medium checked before and after inspissation. If pH instability
is noticed between media or before and after inspissation, this has to be corrected by
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adjusting the buffering capacity of phosphate salt.
MIC distribution for kanamycin
80
70
Number of cases
60
ALL
50
PS
40
PR
30
MDR
non-MDR
20
10
0
5
10
20
30
40
60
80
120
>120
MIC
Preliminary data only: PR and PS not yet completely resolved
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MIC distribution for capreomycin
80
70
No. of cases
60
ALL
50
PS
40
PR
30
MDR
non-MDR
20
10
0
5
10
20
30
40
50
60
80
120
>120
MIC
Preliminary data only: PR and PS not yet completely resolved
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MIC distribution for cycloserine
80
70
No. of cases
60
ALL
50
PS
40
PR
30
MDR
non-MDR
20
10
0
10
20
30
40
50
60
80
120
>120
MIC
Preliminary data only: PR and PS not yet completely resolved
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MIC distribution for ethionamide
50
45
No. of cases
40
35
ALL
30
PS
25
PR
20
MDR
15
non-MDR
10
5
0
5
10
20
30
40
50
60
80
120
>120
MIC
Preliminary data only: PR and PS not yet completely resolved
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MIC distribution for ofloxacin
70
No. of cases
60
50
ALL
40
PS
PR
30
MDR
non-MDR
20
10
0
0.25
0.5
1
2
3
4
6
8
12
>12
MIC
Preliminary data only: PR and PS not yet completely resolved
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MIC distribution for PAS
60
No. of cases
50
ALL
40
PS
30
PR
MDR
20
non-MDR
10
0
0.125 0.25
0.5
1
2
3
4
6
10
>10
MIC
Preliminary data only: PR and PS not yet completely resolved
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What is needed for standardizing
second line drugs testing?
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Thank you
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