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Identification of AFB Directly from VersaTREK ® Myco Bottles by HPLC

Identification of AFB Directly from VersaTREK ® Myco Bottles by HPLC. Vincent LaBombardi, Raxa Katariwala, Giuseppe Pipia St Vincent’s Hospital New York, NY USA. Tuberculosis.

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Identification of AFB Directly from VersaTREK ® Myco Bottles by HPLC

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  1. Identification of AFB Directly from VersaTREK® Myco Bottles by HPLC Vincent LaBombardi, Raxa Katariwala, Giuseppe Pipia St Vincent’s Hospital New York, NY USA

  2. 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 world’s population is currently infected with TB. WHO Tuberculosis Fact Sheet, March 2004.

  3. TB Incidence 2002

  4. TB Incidence 2002

  5. 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

  6. 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 Isolates 2004 SVH-NYC

  7. Infections Caused by NTM • Chronic Pulmonary Disease • Disseminated Disease • Lymphadenitis • Post-Surgical Infections • Endocarditis • Skin and Soft Tissue • Bone and Joint

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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 >.500 with a >.200 separation between first two choices

  13. Chromatographic report for M. tuberculosis Internal Standard # 2 Internal Standard # 1

  14. M. tuberculosis Complex Current Chromatogram Comparison to Reference Chromatogram Reference Chromatogram

  15. M. avium Complex MAC Current Chromatogram Reference Chromatogram

  16. ID from Solid Media

  17. ID from Solid Media

  18. ID from VT Myco Bottles

  19. ID from VT Myco Bottles

  20. TAT Smear Pos-M.tb

  21. TAT Smear Neg-M.tb

  22. 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.

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