Identification of AFB Directly from VersaTREK

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Identification of AFB Directly from VersaTREK
Myco Bottles by HPLC

• Vincent LaBombardi, Raxa Katariwala, Giuseppe
  Pipia
• St Vincents Hospital
• New York, NY USA

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Tuberculosis

• Between 2000 and 2020 nearly 1 billion people
  will become newly infected, 200 million will get
  sick and 35 million will die
• There were 2 million deaths from TB in 2002
• Someone in the world is newly infected with TB
  every second
• Approximately one-third of the worlds population
  is currently infected with TB.
• WHO Tuberculosis
  Fact Sheet, March 2004.

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TB Incidence 2002
Region Cases/100,000 Deaths/100,000
Africa 350 83
The Americas 43 6
Europe 54 8
South-East Asia 182 39
Global 141 29
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TB Incidence 2002
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Other Mycobacterial Species

• Atypical Mycobacteria
• MOTT- Mycobacteria other than Tuberculosis
• NTM- Non-tuberculous Mycobacteria
• PPEM- Potentially Pathogenic Environmental
  Mycobacteria
• Group consists of pathogens, opportunistic
• pathogens, colonizers, and contaminants

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Isolates 2004 SVH-NYC

• M. avium complex 207
• M. tuberculosis 39
• M. abscessus 38
• M. chelonae 17
• M. xenopi 9
• M. gordonae 7
• M. interjectum 6
• M. fortuitum 4

• M. scrofulaceum 3
• M. marinum 3
• M. terrae 2
• M. kansasii 1
• M. simiae 1
• M. mucogenicum 1
• M. thermoresistible 1

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Infections Caused by NTM

• Chronic Pulmonary Disease
• Disseminated Disease
• Lymphadenitis
• Post-Surgical Infections
• Endocarditis
• Skin and Soft Tissue
• Bone and Joint

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Conventional vs. Automated Culture Methods

• Mycobacterium tuberculosis can take anywhere from
  2 to 6-8 weeks to grow on solid media.
• The recovery rates for mycobacteria are higher in
  broth based media than on solid media.
• If susceptibility tests are preformed on solid
  media, the TAT from specimen receipt to final ID
  and susceptibility results can be 2 to 4 months.

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VersaTREK Myco System

• Myco Bottles (12.5 ml 7H 9 Broth)
• Antibiotic Supplement
• AS (Azlocillin, Ampho B, Poly B, Nalidixic acid
• PVNA ( Poly B, Vanco, Nalidixic acid,
  Amphotericin B
• Growth Supplement
• Connectors

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VersaTREK Myco

• Monitors bottle headspace for changes in gas
  pressure
• Looks for a change in slope over three readings
• Mycobacteria will consume oxygen prior to
  producing CO2. Gas pressure in the bottle
  headspace will decrease causing that location to
  signal positive.
• Remove from instrument, stain, perform direct ID

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Direct Testing by HPLC

• ID based on mycolic acid content in the
  mycobacterial cell wall.
• Most species have a unique profile, but some can
  be identified only to complex (M. tuberculosis).
• ID by database analysis takes this technique out
  of the reference lab into the clinical lab.

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Identification Criteria

• Calibration Standard used to provide reference
  retention times and calculate Equivalent Carbon
  Length values ECL).
• Compared to a database
• Generates an ID with a similarity index
• To be acceptable the SI must be gt.500 with a
  gt.200 separation between first two choices

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Chromatographic report for M. tuberculosis
Internal Standard 2
Internal Standard 1
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M. tuberculosis Complex
Current Chromatogram
Reference Chromatogram

• Comparison to Reference Chromatogram

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M. avium Complex
Current Chromatogram
MAC
Reference Chromatogram
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ID from Solid Media
Isolate Test L 1.02 Cor L 1.03 Cor
M. abscessus 9 9 9
M. avium complex 25 20 25
M. celatum 3 3 3
M. chelonae 3 3 3
M. haemophilum 8 7 7
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ID from Solid Media
Isolate Test L 1.02 Cor L 1.03 Cor
M. kansasii 2 2 2
M. scrofulaceum 16 0 15
M. tuberculosis 4 4 4
M. xenopi 6 5 5
Total (19 species) 90 61 (67.8) 82 (91.1)
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ID from VT Myco Bottles
Isolate Test L 1.02 Cor L 1.03 Cor
M. abscessus 6 6 6
M. avium complex 81 16 62
M. celatum 2 2 2
M. chelonae 9 9 9
M. gordonae 9 9 9
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ID from VT Myco Bottles
Isolate Test L 1.02 Cor L 1.03 Cor
M. kansasii 2 2 2
M. scrofulaceum 18 0 15
M. tuberculosis 20 20 20
M. xenopi 6 6 6
Total (19 species) 161 73 (45.3) 135 (83.9)
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TAT Smear Pos-M.tb
N9 Time to Detection Time to ID Suscept Res Avail
Range (Days) 6-20 7-23 14-37
Mean (Days) 10.9 12.4 20.7
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TAT Smear Neg-M.tb
N11 Time to Detection Time to ID Suscept Res Avail
Range (Days) 11-15 12-26 19-39
Mean (Days) 17.3 19.1 28.5
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Conclusions

• The mycolic acid profiles of mycobacteria are
  media dependent.
• The MIDI USFDA cleared database is not adequate
  for identifying isolates directly from VersaTREK
  Myco bottles.
• The re-engineered library has made the
  identification of isolates directly from VT Myco
  bottles feasible and results can be obtained in
  clinically relevant timeframes.