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).
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TB Reference Laboratory,
Department of Health,
BIOSAFETY LEVEL1 2 3 4
Isolationa of laboratory No No Yes Yes
Room sealable for decontamination No No Yes Yes
Ventilation: — inward airflow No Desirable Yes Yes
— controlled ventilating system No Desirable Yes Yes
— HEPA-filtered air exhaust No No Yes/Nob Yes
Double-door entry No No Yes Yes
Airlock No No No Yes
Airlock with shower No No No Yes
Anteroom No No Yes —
Anteroom with shower No No Yes/Noc No
Effluent treatment No No Yes/Noc Yes
Autoclave: — on site No Desirable Yes Yes
— in laboratory room No No Desirable Yes
— double-ended No No Desirable Yes
Biological safety cabinets No Desirable Yes Yes
Personnel safety monitoring capabilityd No No Desirable Yes
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.
WHO Biosafety Manual, 2004
What is the purpose of standardizing second line anti-TB drugs testing?__________________________________________(1) Can clinical relevance of criteria of resistance be ensured?(2) Can reliablelaboratory test procedures be found?(3) Can inter-laboratory comparison be facilitated?
2. Careful selection of laboratories with capability, interest, and commitment, <and can communicate well with clinicians and with each other>.
5 drugs testing?. 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.
6 drugs testing?. 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.
7 drugs testing?. Outcomeis to develop and elaborate a standardized laboratory test protocol using available techniques that can be used under program settings.
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>
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.
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.
(3) Subculture and storage of the strains: drugs testing?
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.
2. Investigation on the drugs testing?in vitro criteria of resistance
Factors that may lead to incorrect laboratory results are:
Failure in inoculum control is usually related drugs testing?to:
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.
M carefully calibrated with clinical isolates of PR and PS MTB, will lead to incorrect resultsany of the second line drugs suffer from inherent difficulties in obtainingreliable resistance criteria
because their MICsare 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.
Metabolic activity of bacilli in the lesions and antibacterial activity of drugs bothdirectly influence the selective growth of drug resistant mutants.
DST results are also greatly affected by physicochemical antibacterial activity of drugs properties of test environment:
DST procedure, mediashould be carefully set up and calibrated to obtain clinically relevant test results taking into account these factors.
OBJECTIVES antibacterial activity of drugs
The project aims to :
Table 2. Anti-TB drugs and their concentrations antibacterial activity of drugs to be tested
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 adjusting the buffering capacity of phosphate salt.