WHO basic standard & infrastructure

requirement of TB laboratory for doing liquid

culture & DST

Somsak Rienthong
NRL & SRL of TB
Bangkok, Thailand

Courtesy: Christopher Gilpin, WHO

 General Recommendations
Culture, DST, TB/HIV

• Clear policy for culture and DST should be

developed based on country situations and
priorities
• The diagnostic process, including all laboratory
tests, should be free of charge
• Access to laboratory tests, such as culture and
DST, should be controlled to prevent misuse.
• Culture and DST should only be expanded in
countries that have adequately implemented
EQA systems for microscopy
 General Recommendations
Culture, DST, TB/HIV

• The budget for laboratory should be earmarked

to prevent transfer for other program areas.
• Establish improved transport for cultures and
specimens
• Explore bulk procurement for equipment (GDF)
• Establish regional subcommittees to help
establish policies for culture and DST to provide
support.
 General Recommendations
Culture, DST, TB/HIV

• Human resource development should be an

essential component of the laboratory
strengthening plan and specifically address
expansion of diagnostics
– Short term needs are training for staff
– Long term training needs should be explored with
academic and other partners to increase technical
capacity
• Quality Assurance
– develop EQA systems for new diagnostics
– Increase support for technical assistance and training
opportunities with regional centers of excellence
Current policy recommendations

• 2007
– Liquid medium for
culture and DST
– Rapid speciation

• 2008
– Line probe assays for
rapid MDR-TB
screening
WHO laboratory policies (1)

• Automated liquid culture and DST (2007): Use of liquid culture systems in the
context of a comprehensive country plan for strengthening TB laboratory capacity;
in a phased manner starting at national/central reference laboratory.

• Rapid speciation (2007): Strip speciation for rapid Mycobacterium tuberculosis

from non-tuberculous mycobacteria; established at regional or central level in
combination with liquid culture.

• Line probe assays (2008): Use of line probe assays for rapid detection of R
resistance within the context of country plans for MDR-TB management, including
development of country-specific screening algorithms and timely access to quality-
assured second-line anti-tuberculosis drugs; do not eliminate the need for
conventional culture and DST capability; should be phased in, starting at
national/central reference laboratory or those with proven molecular capability.

• Second-line drug susceptibility testing (2008): Reliable and reproducible for

injectables and fluoroquinolones; to be conducted in national/central reference
laboratories using standardised methodology and drug concentrations; routine DST
not recommended for ethionamide, prothionamide, cycloserine, terizidone, PAS,
thioacetazone, clofazimine, amoxicillin/clavulanat, clarithromycin, linezolid

Available at: http://www.who.int/tb/dots/laboratory/policy/en/print.html

WHO laboratory policies (2)

• LED microscopy (2010): LED microscopy to replace conventional fluorescent microscopy and
be phased in as replacement for ZN microscopy

• MODS (2010): Recommended for rapid detection of R resistance within the context of country
plans for MDR-TB management, including development of country-specific screening
algorithms and timely access to quality-assured second-line anti-tuberculosis drugs; do not
eliminate the need for conventional culture and DST capability; should be phased in, starting at
national/central reference laboratory, under strict laboratory protocols and quality assurance

• NRA (2010): Recommended as direct or indirect tests, for screening of patients suspected of
having MDR-TB, and acknowledging that time to detection of MDR-TB in indirect application
would not be faster than conventional DST methods using solid culture; to be used within the
context of country plans for MDR-TB management, including development of country-specific
screening algorithms and timely access to quality-assured second-line anti-tuberculosis drugs;
do not eliminate the need for conventional culture and DST capability; should be phased in,
starting at national/central reference laboratory, under strict laboratory protocols and quality
assurance

• CRI methods (2010): Recommended as indirect tests, for screening of patients suspected of
having MDR-TB, and acknowledging that time to detection of MDR-TB would not be faster than
conventional DST methods using liquid culture; to be used within the context of country plans
for MDR-TB management, including development of country-specific screening algorithms and
timely access to quality-assured second-line anti-tuberculosis drugs; do not eliminate the need
for conventional culture and DST capability; should be phased in, starting at national/central
reference laboratory, under strict laboratory protocols and quality assurance

Available at: http://www.who.int/tb/dots/laboratory/policy/en/print.html

Policy framework at country level

• Local epidemiology (TB, HIV, MDR-TB)

• Priorities for case detection
• Local laboratory capacity and networks
• Local laboratory human resources and
skills base
• Local treatment policies for MDR-TB
• Financial resources
Analytical process

• Quantify or estimate TB, TB-HIV and MDR-TB burden

• Identify and target specific patient risk groups
• Quantify or estimate diagnostic tests to identify risk
groups
• Number of suspects to be screened
• Number and type of laboratories at each service level
• Estimate budget for comprehensive laboratory services
• All core components
• Capacity for diagnostic and monitoring
• Ancillary laboratory services (eg. biochemistry, haematology)
Phased approach

• Phase 1: Laboratory preparedness

– Assessment of TB laboratory networks and diagnostic policies
– Upgrade of laboratory infrastructure and biosafety
– Development and implementation of GLP, SOPS, QA, etc.
– Training of core laboratory staff
– Initiating NTP policy reform on diagnostics
• Phase 2: Introduction of new diagnostics
– Integration of new diagnostics into NTP policies and procedures
– Procurement and installation of instruments, reagents, and other
essential supplies
– Validation of new tools and laboratory performance
• Phase 3: Impact assessment
– Continued mentoring, technical support and oversight
– Assessment of impact of new diagnostics
Laboratory algorithm

Starts with
• Screening strategies for suspects
• Microscopy services as entry point
 MICROSCOPY
AFB
(ZN or Fluorescence)
District

Positive Negative No result

CULTURE
(Solid or Liquid)
NRL/regional

Positive Negative No result

TB/NTM
Drug resistance
DRUG SUSCEPTIBILITY TESTING-1st LINE IDENTIFICATION (SPECIATION)
(Solid or Liquid) (Conventional/Commercial)

MDR Not MDR, resistant other drugs Susceptible No result

SRL/NRL

DRUG SUSCEPTIBILITY TESTING-2 nd LINE

(Solid or liquid)

XDR Not XDR, resistant other drugs Susceptible No result

 MICROSCOPY AFB
(ZN or Fluorescence)
District

Positive Negative No result

CULTURE TB/NTM
LINE PROBE ASSAY (Solid or Liquid)
NRL/regional

Positive Negative or no result No result

Drug resistance

DRUG SUSCEPTIBILITY TESTING-1st LINE IDENTIFICATION (SPECIATION)

(Solid or Liquid) (Conventional/Commercial)

MDR Not MDR, resistant other drugs Susceptible No result

SRL/NRL

DRUG SUSCEPTIBILITY TESTING-2 nd LINE

(Solid or liquid)

XDR Not XDR, resistant other drugs Susceptible No result

Policy considerations

• Current technologies not mutually exclusive

– Conventional culture capacity required for SM- specimens
– Conventional DST capacity required to detect XDR-TB
• Liquid culture and line probe assay considered as
gold standards, to be phased in without loss of
existing solid culture and DST capacity
• LED microscopy as alternative for both fluorescence
and conventional light microscopy
• Selected non-commercial culture and DST methods
not alternatives for gold standards, but may provide
interim solution
BSL1
MICROSCOPY
AFB
LED MICROSCOPY
District (ZN or Fluorescence)

Positive Negative or no result

NRA CULTURE
(Solid or Liquid) TB/NTM
LINE PROBE ASSAY
MODS Drug resistance
NRL/regional

Positive Negative or no result

DRUG SUSCEPTIBILITY TESTING-1st LINE IDENTIFICATION (SPECIATION)

(Solid or Liquid) (Conventional/Commercial)
CRI

MDR Not MDR, resistant other drugs Susceptible No result

SRL/NRL

DRUG SUSCEPTIBILITY TESTING-2 nd LINE

(Solid or liquid)
BSL3

XDR Not XDR, resistant other drugs Susceptible No result

MDR-TB diagnosis using solid culture and DST
Microscopy Solid culture 1st line DST 2nd line DST
24h 6-8w 3-4w 3-4w
MDR-TB diagnosis
after 9 to 12 weeks

MDR-TB diagnosis using liquid culture and DST

Microscopy Liquid culture 1st line DST 2nd line DST
24h 2-3w 1-3w 1-2w
MDR-TB diagnosis
after 3 to 5 weeks

MDR-TB diagnosis using line probe assay, liquid culture and DST
Line probe assay
24h
+ MDR-TB
after 1 to
diagnosis
2nd line DST
2 days
1-2w
Microscopy
24h Line probe assay
24h
- Liquid
1st line culture
1-2w
DST
2-3w
1st line DST
2nd
1-3w
1-2w
MDR-TB diagnosis
after 3 to 5 weeks
MDR-TB diagnosis using solid culture and DST
2nd line DST
Microscopy Solid culture 1st line DST 3-4w

24h 6-8w 3-4w

MDR-TB diagnosis
after 9 to 12 weeks

Liquid culture 2nd line DST

2-3w 1-2w
MDR-TB diagnosis using NRA

NRA direct MDR-TB diagnosis

Microscopy
24h
+ 6-9d after 6 to 9 days
NRA direct 2nd line DST
Microscopy
24h
- Solid culture
6-9 days
6-8w
NRA direct
7-21d
1-2w

NRA
1st lineindirect
DST 1st line DST
2nd
1-2w MDR-TB1-2w diagnosis
1-3w
after 7 to 11 weeks
MDR-TB diagnosis using liquid culture and DST
2nd line DST
Microscopy Liquidculture
Liquid culture 1st line DST 3-4w

24h 2-3w
6-8w 3-4w

MDR-TB diagnosis
after 5 to 7 weeks

Liquid culture 2nd line DST

2-3w 1-2w
MDR-TB diagnosis using MODS

Microscopy MODS direct MDR-TB diagnosis

24h
+ 2-21d after 2 to 21 days
NRA direct 2nd line DST
Microscopy
24h
- Solid
Liquid culture
culture
6-9 days
6-8w
2-3w
MODS indirect
6-9d
1-2w

NRA
1st lineindirect
DST 1st line DST
2nd
1-2w MDR-TB1-2w diagnosis
1-3w
after 3 to 4 weeks
XDR-TB diagnosis using conventional solid culture and DST
Microscopy Solid culture 1st line DST* 2ndline
2nd lineDST*
DST
24h 6-8w 3-4w 3-4w
3-4w

XDR-TB diagnosis
* Methods not validated or standardised
after 12 to 16 weeks
XDR-TB diagnosis using liquid culture and DST
Microscopy Liquid culture 1st line DST 2nd
2nd line
line DST
DST
24h 2-3w 1-3w 1-3w
1-2w
XDR-TB diagnosis
after 4 to 9 weeks
XDR-TB diagnosis using line probe assay, liquid culture and DST
2nd line DST
LPA
24h
+ Liquid culture
2-3w
2nd
2nd line
line
1-3w
1-3w
1-2w
DST
DST

Microscopy
24h LPA
24h
- Liquid culture
2-3w
1st 1st
lineline
DST
1-3w
1-2w
DST 2nd
2nd line
line DST
1-3w
1-2w
DST

XDR-TB diagnosis
after 4 to 9 weeks
 Why is Biosafety Needed in
the Mycobacteriology
Laboratory?
• Risk of infection with Mycobacterium tuberculosis is
3-9x higher for TB lab workers than for other lab
workers
• Infection usually results from unrecognized
production of infectious aerosols containing tubercle
bacilli
• Infection can occur from needle sticks, through
broken skin, etc.
 Biosafety

The application of a combination of

administrative controls, containment
principles, laboratory practices and
procedures, safety equipment, and
laboratory facilities to enable
laboratorians to work safely with
potentially infectious microorganisms.
 Administrative Controls
• Supervision by an experienced scientist
• All personnel are well trained, proficient, aware
of hazards, follow rules
• Routine medical surveillance
• Biosafety and operations manuals
• Emergency plans for spills, accidents, etc.
• Appropriate facilities and safety equipment
 Good Laboratory Practices
• Restrict or limit access when working
• Biohazard warning signs
• Prohibit eating, drinking and smoking
• Prohibit mouth pipetting
• Minimize splashes and aerosols
• Decontaminate work surfaces daily
• Decontaminate wastes
 Containment

• Primary Containment: protect worker and

immediate laboratory environment
– good microbiologic techniques
– safety equipment
• Secondary Containment: protect the
environment outside the laboratory
– facility design
– waste management
 Biosafety Level (BSL)

• Conditions under which an infectious agent

can ordinarily be safely handled.
• Conditions are a combination of:
– laboratory practices and techniques
– safety equipment
– laboratory facilities
• Usually agent and procedure specific
 Determining the BSL to Use
• The laboratory director assesses potential risks
for work with a specific agent and assigns a BSL
• Recommended BSLs for many of the infectious
agents have been developed
• Lab directors may specify more or less stringent
practices when information is available to
suggest altered risk
– e.g., increased BSL for XDR TB cultures
– e.g., decreased BSL for the BCG vaccine
 Risk Assessment
• Pathogenicity of the infectious agent
• Route of transmission
• Agent stability and infectious dose
• Concentration of agent
• Type of laboratory procedures to be done
• Availability of effective prophylaxis or therapy
• Skill level and vulnerability of at-risk
personnel
 Biosafety Level 2 (BSL2)
• Suitable for work involving agents of
moderate potential hazard to personnel and
the environment
– Mycobacterium species other than members of
the M. tuberculosis complex
– non-aerosol generating manipulations of clinical
specimens from TB patients
• BSC is to be used for aerosol generating
procedures
 BSL2 – Microbiologic Practices
• Restrict or limit access when working
• Biohazard warning signs
• Prohibit eating, drinking and smoking
• Prohibit mouth pipetting
• Minimize splashes and aerosols
• Decontaminate work surfaces daily
• Decontaminate wastes
 BSL2 – Primary Containment

• Protective clothing - lab coat, gloves, eye

protection
• Class II Biosafety Cabinet used for
manipulations that generate splashes or
aerosols
 BSL2 – Secondary Containment
• Laboratories have lockable doors and are
separated from public areas
• Air flows into lab without re-circulation to non-lab
areas
• Sink for hand washing
• Impermeable, easily cleaned work surfaces
• Eyewash readily available
• Autoclave available
• Windows fitted with flyscreens
 Biosafety Level 3 (BSL3)

• Suitable for work with infectious agents

which may cause serious or potentially
lethal disease as a result of exposure by the
inhalation route.
– members of the M. tuberculosis complex
 BSL3 – Microbiologic Practices
BSL2 practices plus:
• Work in a certified biosafety cabinet
– certified at least annually
• Use bioaerosol-containing equipment
– aerosol-containment centrifuge rotors
• Decontaminate spills promptly
 BSL3 – Primary Containment
• Protective clothing, gloves, eye protection
• Respiratory protection as needed
– N95 respirator or equivalent
– Powered Air-Purifying Respirator (PAPR)
• Class II Biosafety Cabinet used for all open
manipulation of agents
BSL3 – Secondary Containment
BSL2 secondary containment plus:
• Controlled access to a separate area
• Double door entry
• Directional inward airflow
• Single-pass air; 6-12 air changes/hour
• Enclosures for aerosol generating equipment
• Room penetrations sealed
• Walls, floors and ceilings are water resistant
for easy cleaning
If a facility does not have all required BSL3
features (e.g. sealed penetrations, solid ceiling),
an acceptable level of safety for conducting
routine procedures, including culture, may be
achieved in a BSL2 facility providing:
• Directional inward airflow is maintained and
exhaust air is discharged to the outside
• Access to the laboratory is restricted when
work is being performed
• The recommendations for BSL3 practices,
procedures, and safety equipment are
rigorously followed
 Administrative Control
Medical Surveillance

• Done in accordance with national rules

• Surveillance conducted
– at start of work in the TB laboratory
– at regular intervals – at least annually
– after any biohazard incident
 Direct AFB Smear Microscopy

Limited risk of generating infectious aerosols

• Work can be done on an open bench
– restricted access to the laboratory
– separate bench for smear-preparation
• Adequately ventilated laboratory
– 6-12 ACH, directional airflow
– Natural or mechanical ventilation
• Proper disposal of infectious material
 Processing Sputum Specimens for
Smear, Culture, Molecular Tests – 1

Risk of generating infectious aerosols during

centrifugation and specimen manipulation
• Laboratories must have restricted access and
be separated from public areas
• Impermeable surfaces for easy cleaning
• Air flows into lab without re-circulation to non-
lab areas (directional airflow)
– 6-12 ACH, passive or mechanical ventilation
– closed windows
• Proper disposal of infectious material
 Processing Sputum Specimens for
Smear, Culture, Molecular Tests – 2
• Class II Biosafety Cabinet used for all open
manipulation of agents
– BSCs must be properly installed and certified at
least annually
– BSC exhaust may be
• ducted to outside using a hard duct or thimble
fitting
• recirculated into the room if assured that the
BSC is functioning properly
• Use aerosol-containment centrifuge rotors
• Double door entry
 Laboratory Layout &
Equipment

• Plan of Culture Laboratory

• Arranging of equipments & materials
• Care & maintenance of essential
equipment
 Plan of Culture laboratory

• Design for reduce risk of infection &

Contamination
• Suitable in Size & shape and work with other
section
• Control of air flow direction & Ventilation
• Should be separate from other laboratory
activities
 Arranging equipment &
materials
• Laboratory contain facilities necessary for
administration and lab management
• Space for storage, register, reports
chemical and reagent
• Area for received specimen
• reading room
• Media preparation room
• Washing room & sterilization room
 Main laboratory area for:
• Smear preparation
• Specimen processing
• Inoculation
• Incubation
• Equipment: pH, centrifuge, refrigerator?
• Isolation area
Care & maintenance of essential
equipment
• BSC: Class II
• Centrifuge: refrigerated with 3000xg
• Incubator: 35oC or 37oC
• Inspissator: 85oC
• Autoclave: 121oC, 15lb, 15 min
• Hot air oven: 180oC
• Water bath: 37oC & 68oC
• Bunsen burner: foot step control
• Glass ware & plastic ware
 Conclusion
• Capacity

• Capability
• Willing
• Team work