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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,

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Presentation on theme: "Identification of AFB Directly from VersaTREK ® Myco Bottles by HPLC Vincent LaBombardi, Raxa Katariwala, Giuseppe Pipia St Vincents Hospital New York,"— Presentation transcript:

1 Identification of AFB Directly from VersaTREK ® Myco Bottles by HPLC Vincent LaBombardi, Raxa Katariwala, Giuseppe Pipia St Vincents 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 worlds population is currently infected with TB. WHO Tuberculosis Fact Sheet, March 2004.

3 TB Incidence 2002 RegionCases/100,000Deaths/100,000 Africa35083 The Americas436 Europe548 South-East Asia18239 Global14129

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 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

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 CO 2. 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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12 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.

13 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

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

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

17 M. avium Complex Current Chromatogram Reference Chromatogram MAC

18 ID from Solid Media Isolate# TestL 1.02 #CorL 1.03 #Cor M. abscessus999 M. avium complex M. celatum333 M. chelonae333 M. haemophilum877

19 ID from Solid Media Isolate# TestL 1.02 #CorL 1.03 #Cor M. kansasii222 M. scrofulaceum16015 M. tuberculosis444 M. xenopi655 Total (19 species)9061 (67.8%)82 (91.1%)

20 ID from VT Myco Bottles Isolate# TestL 1.02 #CorL 1.03 #Cor M. abscessus666 M. avium complex M. celatum222 M. chelonae999 M. gordonae999

21 ID from VT Myco Bottles Isolate# TestL 1.02 #CorL 1.03 #Cor M. kansasii222 M. scrofulaceum18015 M. tuberculosis20 M. xenopi666 Total (19 species)16173 (45.3%)135 (83.9%)

22 TAT Smear Pos-M.tb N=9Time to Detection Time to IDSuscept Res Avail Range (Days) Mean (Days)

23 TAT Smear Neg-M.tb N=11Time to Detection Time to IDSuscept Res Avail Range (Days) Mean (Days)

24 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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