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Increasing transmission and antimicrobic resistance of Mtbc

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Increasing transmission and antimicrobic resistance of Mtbc & introduction of new technologies require adjustments in laboratory services Safety in the Mycobacteria ... – PowerPoint PPT presentation

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Title: Increasing transmission and antimicrobic resistance of Mtbc


1
Mycobacterial Services
  • Increasing transmission and antimicrobic
    resistance of Mtbc
  • introduction of new technologies require
    adjustments in laboratory services

2
Safety in the Mycobacteria Lab
  • Active TB in Mycobacteriologists
  • 100 x General Population
  • 3 x Other Laboratory Workers
  • Skin Test Conversions in TB Labs
  • 8-30

3
Safety in the Mycobacteria Lab (continued)
  • Non-behavioral risk factors for infection
  • Poor directional airflow
  • Equipment failure
  • Biological safety cabinets
  • Centrifuges
  • Autoclaves
  • Specimens
  • Number of positives processed
  • Organism concentration

4
Safety in the Mycobacteria Lab (continued)
  • Behavioral risk factors for infection
  • Lack of adequate safety guidelines
  • Noncompliance with guidelines
  • Aerosol production
  • Pouring, pipetting mixing liquids
  • Homogenizing cutting tissues
  • Dropping or spilling culture flasks
  • Preparing smears
  • Needle sticks

5
Laboratory Biosafety Levels
  • Biosafety Level - 2
  • Personnel trained in handling pathogens
  • Lab directed by competent scientist
  • Access to lab is limited during work periods
  • Extreme precautions with sharps
  • Aerosol-producing procedures conducted in
    biologic safety cabinet

6
Laboratory Biosafety Levels (continued)
  • Biosafety Level - 3
  • Training in handling agents acquired by
    inhalation able to cause lethal disease
  • Supervised by scientists experienced in working
    with such agents
  • P3 laboratory facilities and procedures

7
Levels of Laboratory ServiceATS Recommendations
  • Level I
  • Collect, smear transport specimens
  • Level II
  • Smear culture ID sensi testing of Mtb
  • Level III
  • Smear culture ID sensi testing of all
    mycobacterial isolates

8
Level I Laboratories
  • Tasks
  • Collect good specimens
  • Prepare examine smears
  • Ship to Level II/III for culture sensitivities
  • Smear proficiency test scores of gt 80

9
Level I Laboratories (continued)
  • Characteristics
  • Receive minimum of 10 AFB smears/week
  • Use fluorochrome stain
  • Quantitate smear AFB report within 24 hr.
  • Use certified biologic safety cabinet
  • Send specimen for culture within 24 hr.
  • Meet Dept. of HHS Biosafety Level 2 (BL-2)
    requirements

10
Sputum Specimen Collection
  • Collection of good specimen is critical
  • First morning expectoration
  • Rinse mouth with water
  • Produce specimen by deep coughing
  • Expectorate directly into lab-provided tube
  • If specimen inadequate, induce sputum
  • Dispatch immediately to lab
  • Containers to be closed and sealed
  • Refrigerate if not processed immediately

11
AFB Smears
  • Level I Laboratories
  • Recommended specimen sterilization
  • Open in BSC sterilize with Clorox x 15 min
  • Stain read on open bench
  • Specimen cannot be used for culture
  • Probably safe - not currently recommended
  • Open, prepare, dry heat fix x 2h in BSC
  • Stain read on open bench
  • Dispatch sealed container to Level II/III lab

12
Level II Laboratories
  • Tasks
  • Perform all procedures done by Level I
  • Isolate organisms in pure culture
  • Identify M. tuberculosis complex
  • Perform susceptibility testing of Mtb
  • Refer MOTT isolates for ID sensitivities
  • Retain recovered isolates for 6 months
  • Proficiency testing of isolation, ID and
    sensitivity procedures

13
Level II Laboratories (continued)
  • Characteristics
  • Receive minimum of 20 specimens/week
  • Use Class II or III biologic safety cabinet
  • Detect growth in liquid media in 10 days
  • ID with DNA probes, BACTEC NAP or mycolic acid
    profile within 2-3 weeks
  • Determine sensis of Mtb isolates in 3-4 w
  • Meet Dept of HHS Biosafety Level 3 (BL-3)
    requirements

14
AFB Stains
  • Concentrated Smear for Stain Culture
  • Done in Level III laboratory BSC
  • Liquify decontaminate
  • Centrifuge gt 3000G x 15min in sealed cups
  • Prepare smear, dry, heat fix x2 h
  • Stain and read within BL-3 facility
  • Flourochrome (auramine) stains preferred

15
Level III Laboratories
  • Tasks
  • Identify all mycobacterium
  • Perform susceptibility tests on MOTTs
  • Proficiency testing on MOTT identifications
  • Work concentrated in very few labs

16
Level III Laboratories (continued)
  • Characteristics
  • Perform 20 susceptibilities per month
  • USE DNA probes, HPLC or other rapid ID methods
  • Do not rely only on NA amplification for ID
  • Meet Dept HHS Biosafety Level 3 (BL-3)
    requirements

17
New Technologies
  • Fluorescent microscopy
  • Radiometric culture systems
  • NAP test, DNA probes, HPLC
  • Radiometric susceptibility testing
  • Direct amplification of mycobacterial nucleic
    acid from specimens

18
New Technologies (continued)Direct Amplification
Techniques
  • AMTD approved for smear respiratory specimens
  • Requires 4-5 hours for completion
  • Detects only M. tuberculosis complex
  • Specificity approaches 100
  • Sensitivity 90-100 less in smear negative
    specimens

19
AFB Stains
  • Sensitivity on expectorated sputum
  • M. tuberculosis 65
  • MOTT 20
  • Cum Sensitivity in series of 3 sputa
  • M. tuberculosis 80

20
AFB Stains
  • Cum sensi of smears in culture series that yield
    both a () culture for M. tuberculosis and one or
    more positive smears
  • First specimen 86
  • Second specimen 95
  • Third specimen 95
  • Fourth specimen 100

21
AFB Stains (continued)
  • Specificity
  • Abs. False () 0.05
  • Rel. False () 1.8
  • Specificity 99.9
  • PPV 98.1

22
Culture
23
Culture (continued)
24
CDC Isolation Guidelines
  • Initiation of Isolation
  • Member of high risk population
  • Contact with infectious TB patient
  • Born in area of high TB prevalence
  • Medically under-served populations
  • HIV, elderly, homeless, imprisoned
  • Alcoholics, IV drug users
  • Appropriate clinical findings

25
CDC Isolation Guidelines (continued)
  • Initiation of Isolation
  • Member of high risk population
  • Appropriate clinical findings
  • Cough gt 3 weeks
  • Bloody sputum,
  • Anorexia, weight loss
  • Fever, night sweats

26
CDC Isolation Guidelines
  • Discontinuation of Isolation
  • Patients with proven infectious TB
  • Appropriate Rx (optimally 2-3w)
  • Clinical Improvement
  • 3 consecutive negative smears (q2w)
  • Pt discharged to suitable locale
  • Patients suspected of having infectious TB
  • Alternate diagnosis confirmed
  • Pt determined to be noninfectious

27
Availability of AFB Smears
  • CDC recommendations
  • Results available within 24 hours
  • No longer refer to STAT smears

28
The End
  • Questions

29
Timeline Comparison of TB MethodologyNew verses
Old
  • Specimen
  • Received
  • Week 0 1
    2 3 4 5
    6 7 8 9 10 11 12 13 14 15
    16 17 18
  • ______________________
    __________________________________________________
    ____________
  • Mycobacteria growth
  • Bactec 460 System
  • TB DNA Probe ID
  • (after growth)
  • Bactec Sensitivities

MTD
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