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