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Identification of AFB Directly from VersaTREK® Myco Bottles by HPLC

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Presentation on theme: "Identification of AFB Directly from VersaTREK® Myco Bottles by HPLC"— Presentation transcript:

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 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

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 M. chelonae M. xenopi M. gordonae M. interjectum M. fortuitum M. scrofulaceum M. marinum M. terrae M. kansasii M. simiae M. mucogenicum M. thermoresistible

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

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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

14

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

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

17 M. avium Complex MAC Current Chromatogram Reference Chromatogram

18 ID from Solid Media Isolate # Test L 1.02 #Cor L 1.03 #Cor
M. abscessus 9 M. avium complex 25 20 M. celatum 3 M. chelonae M. haemophilum 8 7

19 ID from Solid Media Isolate # Test L 1.02 #Cor L 1.03 #Cor M. kansasii
M. scrofulaceum 16 15 M. tuberculosis 4 M. xenopi 6 5 Total (19 species) 90 61 (67.8%) 82 (91.1%)

20 ID from VT Myco Bottles Isolate # Test L 1.02 #Cor L 1.03 #Cor
M. abscessus 6 M. avium complex 81 16 62 M. celatum 2 M. chelonae 9 M. gordonae

21 ID from VT Myco Bottles Isolate # Test L 1.02 #Cor L 1.03 #Cor
M. kansasii 2 M. scrofulaceum 18 15 M. tuberculosis 20 M. xenopi 6 Total (19 species) 161 73 (45.3%) 135 (83.9%)

22 TAT Smear Pos-M.tb N=9 Time to Detection Time to ID Suscept Res Avail
Range (Days) 6-20 7-23 14-37 Mean 10.9 12.4 20.7

23 TAT Smear Neg-M.tb N=11 Time to Detection Time to ID Suscept Res Avail
Range (Days) 11-15 12-26 19-39 Mean 17.3 19.1 28.5

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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