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 PipiaSt Vincent’s HospitalNew York, NY USA
2 TuberculosisBetween 2000 and 2020 nearly 1 billion people will become newly infected, 200 million will get sick and 35 million will dieThere were 2 million deaths from TB in 2002Someone in the world is newly infected with TB every secondApproximately 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 Americas436Europe548South-East Asia18239Global14129
5 Other Mycobacterial Species “Atypical” MycobacteriaMOTT- Mycobacteria other than TuberculosisNTM- Non-tuberculous MycobacteriaPPEM- Potentially Pathogenic Environmental MycobacteriaGroup consists of pathogens, opportunisticpathogens, colonizers, and contaminants
6 Isolates 2004 SVH-NYC M. avium complex 207 M. tuberculosis 39 M. abscessusM. chelonaeM. xenopiM. gordonaeM. interjectumM. fortuitumM. scrofulaceumM. marinumM. terraeM. kansasiiM. simiaeM. mucogenicumM. thermoresistible
7 Infections Caused by NTM Chronic Pulmonary DiseaseDisseminated DiseaseLymphadenitisPost-Surgical InfectionsEndocarditisSkin and Soft TissueBone 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.
10 VersaTREK Myco Monitors bottle headspace for changes in gas pressure Looks for a change in slope over three readingsMycobacteria 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
12 Direct Testing by HPLCID 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 databaseGenerates an ID with a similarity indexTo be acceptable the SI must be >.500 with a >.200 separation between first two choices
15 Chromatographic report for M. tuberculosis Internal Standard # 2Internal Standard # 1
16 M. tuberculosis Complex Current ChromatogramReference ChromatogramComparison to Reference Chromatogram
17 M. avium ComplexMACCurrent ChromatogramReference Chromatogram
18 ID from Solid Media Isolate # Test L 1.02 #Cor L 1.03 #Cor M. abscessus9M. avium complex2520M. celatum3M. chelonaeM. haemophilum87
19 ID from Solid Media Isolate # Test L 1.02 #Cor L 1.03 #Cor M. kansasii M. scrofulaceum1615M. tuberculosis4M. xenopi65Total (19 species)9061 (67.8%)82 (91.1%)
20 ID from VT Myco Bottles Isolate # Test L 1.02 #Cor L 1.03 #Cor M. abscessus6M. avium complex811662M. celatum2M. chelonae9M. gordonae
21 ID from VT Myco Bottles Isolate # Test L 1.02 #Cor L 1.03 #Cor M. kansasii2M. scrofulaceum1815M. tuberculosis20M. xenopi6Total (19 species)16173 (45.3%)135 (83.9%)
22 TAT Smear Pos-M.tb N=9 Time to Detection Time to ID Suscept Res Avail Range(Days)6-207-2314-37Mean10.912.420.7
23 TAT Smear Neg-M.tb N=11 Time to Detection Time to ID Suscept Res Avail Range(Days)11-1512-2619-39Mean17.319.128.5
24 ConclusionsThe 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.