Identification of AFB Directly Identification of AFB Directly from VersaTREKfrom VersaTREK®® Myco Bottles Myco Bottles

by HPLCby HPLC

Vincent LaBombardi, Raxa Katariwala, Giuseppe Pipia

St Vincent’s Hospital

New York, NY USA

TuberculosisTuberculosis

• 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 2002TB 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

TB Incidence 2002TB Incidence 2002

Other Mycobacterial SpeciesOther 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-NYCIsolates 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

Infections Caused by NTMInfections Caused by NTM

• Chronic Pulmonary Disease• Disseminated Disease• Lymphadenitis• Post-Surgical Infections• Endocarditis• Skin and Soft Tissue• Bone and Joint

Conventional vs. Automated Conventional vs. Automated Culture MethodsCulture 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 SystemVersaTREK 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 MycoVersaTREK 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

Direct Testing by HPLCDirect 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 CriteriaIdentification 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

Chromatographic report for Chromatographic report for M. tuberculosisM. tuberculosis

min3 4 5 6 7 8 9 10

LU

0

50

100

150

200

250

300

FLD1 A, Ex=345, Em=425 (C:\SHERLOCK\RAW\E02423.498\A0082057.D)

2.581 2.750 2.914

3.086 5.803 6.152 6.453

6.664

6.945

7.219

7.494

7.775

8.042

8.310

8.549

8.833

9.305 9.529 9.724 9.915

Internal Standard # 1

Internal Standard # 2

M. tuberculosisM. tuberculosis Complex Complex

Current Chromatogram

Reference Chromatogram

Comparison to Reference Chromatogram

M. aviumM. avium Complex Complex

Current Chromatogram

Reference Chromatogram

MAC

ID from Solid MediaID from Solid Media

Isolate # Test L 1.02 #Cor L 1.03 #Cor

M. abscessus 9 9 9

M. avium complex 25 20 25

M. celatum 3 3 3

M. chelonae 3 3 3

M. haemophilum 8 7 7

ID from Solid MediaID from Solid Media

Isolate # Test L 1.02 #Cor L 1.03 #Cor

M. kansasii 2 2 2

M. scrofulaceum 16 0 15

M. tuberculosis 4 4 4

M. xenopi 6 5 5

Total (19 species) 90 61 (67.8%) 82 (91.1%)

ID from VT Myco BottlesID from VT Myco Bottles

Isolate # Test L 1.02 #Cor L 1.03 #Cor

M. abscessus 6 6 6

M. avium complex

81 16 62

M. celatum 2 2 2

M. chelonae 9 9 9

M. gordonae 9 9 9

ID from VT Myco BottlesID from VT Myco Bottles

Isolate # Test L 1.02 #Cor L 1.03 #Cor

M. kansasii 2 2 2

M. scrofulaceum 18 0 15

M. tuberculosis 20 20 20

M. xenopi 6 6 6

Total (19 species) 161 73 (45.3%) 135 (83.9%)

TAT Smear Pos-TAT Smear Pos-M.tbM.tb

N=9 Time to Detection

Time to ID Suscept Res Avail

Range

(Days)

6-20 7-23 14-37

Mean

(Days)

10.9 12.4 20.7

TAT Smear Neg-TAT Smear Neg-M.tbM.tb

N=11 Time to Detection

Time to ID Suscept Res Avail

Range

(Days)

11-15 12-26 19-39

Mean

(Days)

17.3 19.1 28.5

ConclusionsConclusions

• 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.