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.

2 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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5 Table 3. Summary of biosafety level requirements BIOSAFETY LEVEL Isolation a 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/No b 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/No c No Effluent treatment No No Yes/No c 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 capability d 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

6 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, …..?

7 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?

8 Organization and Planning Prior to calibration of the DST testing methods: 1.Careful selection of TB institutes or TB hospitals, that (i)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,.

9 3.Representative sample of M. tuberculosis strains (i)Most important issue; (ii)Well-documented; (iii)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.

10 4. Requirements for quality control a.Internal quality control – media, H37RV, drug concentrations b.External quality control

11 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.

12 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.

13 7. Outcome is to develop and elaborate a standardized laboratory test protocol using available techniques that can be used under program settings.

14 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.

15 (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.

16 (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.

17 (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.

18 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.

19 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.

20 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.

21 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.

22 Metabolic activity of bacilli in the lesions and antibacterial activity of drugs both directly influence the selective growth of drug resistant mutants.

23 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.

24 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.

25 Table 2. Anti-TB drugs and their concentrations to be tested Media Drug concentrations ( ㎍ / ㎖ ) to be tested KMCPMPTHCSOFXPAS L-J Medium 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.

26 Preliminary data only: PR and PS not yet completely resolved

27 Preliminary data only: PR and PS not yet completely resolved

28 Preliminary data only: PR and PS not yet completely resolved

29 Preliminary data only: PR and PS not yet completely resolved

30 Preliminary data only: PR and PS not yet completely resolved

31 Preliminary data only: PR and PS not yet completely resolved

32 What is needed for standardizing second line drugs testing?

33 Thank you