Transcript Automation in Blood and Mycobacterial Cultures
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
• • • •
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.
TB Incidence 2002 Region Africa The Americas Europe South-East Asia Global Cases/100,000 Deaths/100,000 350 83 43 54 6 8 182 141 39 29
TB Incidence 2002
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
Isolates 2004 SVH-NYC M. avium complex 207
M. tuberculosis
39
M. abscessus M. chelonae
38 17
M. xenopi M. gordonae M. interjectum M. fortuitum
9 7 6 4
M. scrofulaceum M. marinum M. terrae M. kansasii M. simiae M. mucogenicum M. thermoresistible
1 3 3 2 1 1 1
Infections Caused by NTM
• • • • • • • Chronic Pulmonary Disease Disseminated Disease Lymphadenitis Post-Surgical Infections Endocarditis Skin and Soft Tissue Bone and Joint
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.
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
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
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
.
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
300
Chromatographic report for
LU
M. tuberculosis
FLD1 A, Ex=345, Em=425 (C:\SHERLOCK\RAW\E02423.498\A0082057.D) 250 200 150 100
Internal Standard # 1
50
Internal Standard # 2
0 3 4 5 6 7 8 9 10 min
M. tuberculosis Complex Current Chromatogram Reference Chromatogram Comparison to Reference Chromatogram
M. avium Complex
MAC
Current Chromatogram Reference Chromatogram
ID from Solid Media Isolate
M. abscessus
M. avium complex
M. celatum M. chelonae M. haemophilum
# Test L 1.02 #Cor L 1.03 #Cor 9 25 9 20 9 25 3 3 8 3 3 7 3 3 7
ID from Solid Media Isolate
M. kansasii M. scrofulaceum M. tuberculosis M. xenopi
Total (19 species) # Test 2 16 4 6 90 L 1.02 #Cor L 1.03 #Cor 2 0 2 15 4 5 61 (67.8%) 4 5 82 (91.1%)
ID from VT Myco Bottles Isolate
M. abscessus M. avium
complex
M. celatum M. chelonae M. gordonae
# Test L 1.02 #Cor L 1.03 #Cor 6 81 6 16 6 62 2 9 9 2 9 9 2 9 9
ID from VT Myco Bottles Isolate
M. kansasii M. scrofulaceum M. tuberculosis M. xenopi
Total (19 species) # Test L 1.02 #Cor L 1.03 #Cor 2 2 2 18 20 0 20 15 20 6 161 6 6 73 (45.3%) 135 (83.9%)
TAT Smear Pos-M.tb N=9 Time to Detection Time to ID Suscept Res Avail 6-20 7-23 14-37 Range (Days) Mean (Days) 10.9
12.4
20.7
TAT Smear Neg-M.tb N=11 Time to Detection Time to ID Suscept Res Avail 11-15 12-26 19-39 Range (Days) Mean (Days) 17.3
19.1
28.5
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.