Laboratory requirements for MDRTB

1
Laboratory requirements for MDR-TB
Training workshop on multi-drug resistance Cairo,
Egypt, 25-29 November 2007
Véronique Vincent WHO, STB, THD
2
(No Transcript)
3
(No Transcript)
4
Phenotypic tests

• Solid medium (egg-based, agar-based)
• Resistance defined by critical proportion of
  growth in drug-containing media
• Proportion method
• Absolute concentration method
• Resistance ratio method
• Liquid medium
• Resistance defined by selective growth in the
  presence of drug
• BACTEC (Becton Dickinson)
• MGIT (Becton Dickinson)
• MBBacT (bioMérieux)
• Bacti-Alert (Organon Technika)

5
Solid versus liquid media
Liquid culture increases sensitivity (smear-) and
reduces delays.
6
Culture on liquid media
Liquid medium Non radioactive
Liquid medium Bactec radiometric
7
Automates for liquid culture
8
First-line DST methods

• Methodology for 1st-line drugs (H, R, S, E)
  well-established and standardised
• Results very similar using different
  methodologies, including liquid medium
• Performance characteristics well-described
• WHO recommendations for the use of liquid
  cultures

9
DST inaccuracy Sources

• Drug powder quality, storage expiry
• Correct drug concentration Potency!
• Drug dissolution and dilution
• Medium preparation (heating) and storage
• Strain viability
• Inoculum size dispersion
• Too early reading
• Neglect to use positive and negative controls
• QA is essential

10
Role of the NRL

• NRL performs DST
• specimens transported to the NRL for culture and
  DST
• if regional culture labs, cultures transported
  to the NRL
• NRL ensures the quality of microscopy / culture/
  DST within the lab network
• establishes a regular on-site supervision
  programme
• provides training and QA for lab procedures
• Critical issue HR, adequate training, adequate
  staffing
• In countries where culture/DST is decentralized,
  DST results have to be QA by the NRL

11
The Supranational Laboratory Network (SRLN) 2006
(links with gt120 countries)
Coordinating Centre
SRL
12
SRLN System of External Quality Assurance
Coordinating Centre Antwerp, Belgium
Panel of 30 coded isolates
Network of 25 Supranational Laboratories
Initial Assessment
Sample of isolates for rechecking (DRS/DOTS-Plus)
Panel of 20 coded isolates
Ongoing TA
National/Regional Reference Laboratories
13
(No Transcript)
14
Aerosol Formation Spread of droplets
Coughing Singing Sneezing Talking
15
Procedural hazards be aware!

• Production of infectious aerosols in the
  laboratory
• Adding decontamination solutions
• Working with loops
• Pipetting
• Pouring suspensions of bacilli
• HANDLING OF LIQUIDS
• Ingestion hazards
• Inoculation hazards

16
TB developed among 7/15 DST technicians compared
to only 2/59 culture/non-DST technicians.
Compared to non-laboratory workers, the relative
risk for DST technicians was 21.5 (95CI
4.5102.5). CONCLUSION DST led to the highest
relative risk among all types of mycobacteriology
work, while performing smear microscopy alone did
not pose an elevated risk compared to clerical
work.
17
Biological Safety Cabinets in the
mycobacteriology laboratory

• The most crucial piece of equipment in all
  diagnostic mycobacteriology laboratories is the
  biological safety cabinet (BSC).

18
What is a Biological SafetyCabinet ?

• A ventilated area providing protection
• of the user and of the environment
• against infectious aerosols
• while handling hazardous micro-organisms

19
HEPA Filter
Glossaire

• H.E.P.A High Efficiency Particule Air
  Filter
• Traps 99.97 of particles of 0,3µm in
  diameter
• Only microbe-free exhaust air is discharged
  from the BSC

20
Biosafety cabinet

• HEPA filter traps 99.97 of particles
• BSCs should be ducted or vented to the outside
• Whatever the type I or II
• No recirculation allowed
• into the room

21
Evaluation of effectiveness
Maintenance PSM type II

• Operations performed by the manufacturer or
  qualified professional, with specific equipment
  well calibrated

22
Performance Testing (at least once a year)

• Down flow Velocity and Volume test
• Inflow Velocity Test
• Airflow Smoke Pattern Test
• HEPA Filter Leak Test
• Cabinet Leak Test
• Electrical Leakage and Ground Circuit Resistaance
  and polarity Test
• Lighting Intensity Test
• Vibration Test
• Noise Level Test
• UV Lamp Test

23
Fumigation
Maintenance PSM type II

• Decontamination of the BSC with formaldehyde
  gas

Système DECONTAKIT
24
Precautions
Installation
A et B avoid D et C recommended
C
D
A
B
25
Biosafety level
Glossaire

• Classification based on 3 notions
• Layout of the laboratory
• Safe equipment
• Laboratory practices

26
(No Transcript)
27
WHO laboratory biosafety manual
28
BSL 3
29
The mycobacteriology laboratory BSL2 or BSL3
facilities ?

• The BSL-2 laboratory area specimen processing
  for culture in a BSC
• The BSL-3 laboratory area culture
  identification, drug susceptibility testing, and
  other tests that require concentrated cell
  suspensions.
• knowledgeable personnel working under close
  supervision.

30

• Pre-requisites for upgrading BSL-3
• For most high burden countries, there are major
  constraints to the successful establishment,
  staffing and maintenance of BSL3 labs.
• Upgrading of BSL3 should be planned, financed
  and implemented according to a short-term plan, a
  responsibility of the country.

31
(No Transcript)
32
(No Transcript)
33
(No Transcript)
34
Objectives of surveillance

• Estimate the magnitude of drug resistance
  globally
• Determine trends
• Evaluate the progress of TB programmes
• Regimen evaluation
• Data to inform policy decisions MDR-TB
  management

35
Principles of Anti-Tuberculosis Drug Resistance
Surveillance

• 1. Sample accurately represents population under
  study
• Representative group of new TB cases
• Representative group of previously treated TB
  cases
• Examples surveillance, 100, cluster, population
  proportionate cluster
• 2. Quality assured laboratory results
• Supranational Laboratory Network
• 25 laboratories, coordinating center, PT and QA
• 3. Differentiation between new and previously
  treated cases
• treatment history
• clinical records

36
SRLN System of External Quality Assurance
Coordinating Centre Antwerp, Belgium
Panel of 30 coded isolates
Network of 25 Supranational Laboratories
Initial Assessment
Sample of isolates for rechecking (DRS/DOTS-Plus)
Panel of 20 coded isolates
Ongoing TA
National/Regional Reference Laboratories
37
Protocol

• Introduction and background
• epi situation, lab network, previous data
• Objectives
• clear and concise
• Methods
• survey design and sampling strategy
• Intake period and logistics
• inclusion criteria, sputum collection, forms,
  transportation
• Laboratory methods
• proportion, absolute concentration, resistance
  ratio
• Quality assurance
• internal, external
• Data management
• data collection, double entry, analysis
• Human resources needed
• principal investigator, team
• Financial resources

38
Policy Guidance on Drug Susceptibility Testing
(DST) of Second-line Anti-Tuberculosis
DrugsDRAFT

• The document provides an interim policy framework
  for the lab component relevant to programmatic
  implementation of MDR TB strategies
• The document addresses
• Drug efficacy of SLD
• Reliability and reproducibility of SLD
• Cross resistance
• DST methodology and critical concentrations for
  SLD

39
SLD DST inaccuracy Sources

• Additional technical difficulties
• In vitro drug instability
• Drug loss due to protein binding
• Heat inactivation
• Incomplete dissolution
• Drug potency
• QA is essential

40
SLD DST inaccuracy Sources

• the drug critical concentration defining
  resistance is often very close to the MIC
  required to achieve antimycobacterial activity,
• increased probability for misclassification of
  susceptibility or resistance and leading to poor
  reproducibility of DST results.
• QA is essential

41
Policy Guidance on Drug Susceptibility Testing
(DST) of Second-line Anti-Tuberculosis
DrugsDRAFT
I Extensive published studies, extensive
multi-centre laboratory review, broad
inter-method agreement, high stability of drug
powder in vitro, consistent DST reliability and
reproducibility, extensive clinical outcome
data HRE II Extensive published studies,
extensive multi-centre laboratory review, limited
inter-method agreement, variable DST
reproducibility (and therefore reliability),
variable stability of drug powder in vitro, less
extensive clinical outcome data ZS Kanamycin
Amikacin Capreomycin III Less extensive
published studies, limited multi-centre
laboratory review, limited inter-method
agreement, limited data on DST reproducibility
and reliability, limited data on drug powder
stability in vitro, limited clinical outcome
data Ciprofloxacin Ofloxacin
42
Policy Guidance on Drug Susceptibility Testing
(DST) of Second-line Anti-Tuberculosis
DrugsDRAFT
IV Limited or no published studies, limited
multi-centre laboratory review, limited data on
questionable DST reproducibility (and therefore
reliability), instability of drug powder in
vitro, no clinical outcome data Ethionamide
Prothionamide Cycloserine PAS Levofloxacin
Moxifloxacin Gatifloxacin V No published
studies, no multi-centre laboratory review,
reproducibility and reliability impossible to
assess, unknown stability of drug powder in
vitro, no clinical outcome data. Thioacetazone
Clofazimine Amoxicillin Clarithromycin Linezolid
43
Policy Guidance on Drug Susceptibility Testing
(DST) of Second-line Anti-Tuberculosis
DrugsDRAFT
Rational use of DST in programmes for control of
drug-resistant TB As a minimum, lab capacity to
reliably detect INH and RIF resistance is a
prerequisite. The use of rapid detection of Rif
resistance is recommended in high risk MDR
settings Routine DST for SLD is not
recommended In order to retain proficiency and
expertise, it is recommended that second-line DST
only be performed if at least 200 specimens from
high-risk patients per year are expected. This
implies centralization of laboratory services for
SLD or outsourcing such services (eg. to one of
the laboratories in the SRL Network) where
programmes involve small numbers of MDR-TB
patients.
44
Policy Guidance on Drug Susceptibility Testing
(DST) of Second-line Anti-Tuberculosis
DrugsDRAFT
Systematic approach to implementation of DST
under routine programmatic conditions Step 1
Isoniazid, Rifampicin Step 2 Ethambutol,
Streptomycin, Pyrazinamide Step 3 Amikacin,
Kanamycin, Capreomycin, fluoroquinolone Steps 1
and 2 may be merged if indicated by
epidemiological considerations and/or treatment
modalities (eg. standardized or individualised
MDR-TB regimens still involving first-line drugs)
and if resources allow extended DST capacity.
Steps 1 and 3 may be merged in settings where
XDR is a concern in order to rapidly allow the
identification of XDR-TB patients.
45
ISSUES in DST and DRS

• Laboratory
• SAFETY of Culture and DST
• AVAILABILITY of Culture and DST
• QUALITY of Culture and DST
• TRANSPORT of strains expensive, legal
  implications
• HUMAN RESOURCE

46
Rapid tests for DST and new diagnostic tools

• Molecular tests
• Rapid few hours
• Detect resistance on alive and dead bacilli
• Less sensitive safety issues
• Limited to detection of RIF INH
• Require specific training/settings
• Phenotypic tests
• Slow
• Detect resistance in viable bacilli
• Require BSL3
• Can be used for all drugs

47
GenoType MTBDRplus test procedure
From Rick O'Brien, FIND Molecular tests for the
detection of resistance to RIF and INH
3) Hybridization Reverse hybridization of
amplified nucleic acids to specific DNA probes
bound on strips

• DNA
• Extraction

2) Amplification by PCR
4) Evaluation
48
Reaction zones of GenoType MTBDRplus (examples)
49
Colorimetric tests(Alamar-blue, MTT Resazurin)

• Redox-indicators ? colour change with oxidation
• MIC, precise for essential drugs
• Inexpensive
• Only from pure culture
• Microtiter plate format, liquid, less safe
• Nitrate reduction detection
• Precise, inexpensive,
• No special safety problem
• Not properly validated

50
Microscopic observation assay

• This assay uses inverted light microscope and
  Middlebrook 7H9 broth medium to detect
  mycobacterial growth.
• In a recent large study in Peru it had a median
  time to culture positivity on average 8
  days.
• It may be used for drug susceptibility also.

MOORE DAJ, et al, Microscopic observation drug
susceptibility assay, a rapid reliable diagnostic
test for MDR suitable for use in resources-poor
setting, J Clin Microbiol 2004, 42 4432 -
4437
51
(No Transcript)
52
New rapid tests

• Rapid phenotypic tests
• Still in development
• Early validation stage or early field
  demonstration phase
• No rapid identification tests available for SLD

53
New rapid tests

• Molecular tests for the detection of resistance
  to RIF (INH) well evaluated on cultures in
  laboratory based studies
• Current evaluation for feasibility, cost
  effectiveness and cost-benefit in the field
• Field testing on samples (FIND studies)
• No WHO recommendation at present
• Should be available next year