Update on MALDI-TOF Validation

Transcription

1 Update on MALDI-TOF Validation Donald Busalacchi B.S. Microbiologist- WSLH WMLN 2015

2 Review Matrix-Assisted Laser Desorption Ionization Time-of- Flight A form of Mass Spectroscopy utilizing a soft ionization technique. Used to analyze proteins and other biomolecules. Separation of such molecules based on mass to charge ratio occurs along TOF tube as molecules travel to detector. Dedicated software analyzes mass spectra against stored profiles and gives microorganism identification.

3 Culture suspension & wash steps Heat lysis Ethanol wash Bead Beat 70% formic acid/ acetonitrile addition

4 Continued validation outline Impact of media type and culture age on MALDI score. Ongoing species validation. Post-extraction stability study. Identification from MGIT broth. Limit of detection Spectral background noise Current WSLH testing algorithm.

5 Culture age/media type Study Four control strains of mycobacteria (TB, MAC, M. chelonae, and M. abscessus) were grown on three media types (7H9 broth, 7H10 plate, and LJ slant). These cultures were extracted and analyzed in duplicate at 7 time points across 42 days of incubation.

6 Effect of Media on MALDI Score 17

7 Effect of Culture Age on MALDI Score 18

8 Culture age/media type Study Media type: 7H9 Broth, 7H10 Plate, and LJ Slant could all be used with no difference in identification and no significant difference in score. Culture age: fresh culture represent the best chance for good MALDI identification. A significant decrease in score over time was observed in solid media cultures of rapidly growing mycobacteria.

9 Species Validation We have validated TB, MAC, and 16 species of NTM for direct identification by MALDI.

10 New Library Improvements Bruker recently released a new iteration of the mycobacteria library(v 3.0) with 21 new species. Now able to better differentiate between members of the M. fortuitum group (M. farcinogense vs. M. sengalense vs. M. porcinum). Now able to identify: M. parascrofulaceum M. goodi M. salminophilum

11 Remaining Discrepancies Cannot distinguish between members of the MTB complex. Cannot distinguish some closely related species. For example: M. shotsii vs. M. marinum M. mucogenicum vs. M. marinum M. kansasii vs. M. gastri Identifies some isolates of M. gordonae as Mycobacteria species(nontuberculous) Cannot Identify M. abscessus to subspecies level.

12 Post extraction stability Three control strains of mycobacteria (TB, MAC, and M. fortuitum) were grown and extracted from two media types(7h10 and 7H9). The extract supernatants were held at room temperature and analyzed at various time points across 45 hours.

13 Post extraction stability

14 Identification from MGIT cultures Goal: effectively identify mycobacteria directly from positive MGIT without waiting for subculture growth. This is not currently our practice. Why? What are the barriers to success? Concentration necessary for identification Interference from primary MGIT broth components.

15 Limit of detection

16 Limit of detection The concentration of cells need for identification from MGIT broth is roughly 6.00x10 8 cells. The approximate concentration at which MGIT tube flags positive is 1 X 10 5 to 1 X 10 6 cells/ml (BD package insert). Therefore, MGITs may require an extended incubation before they can be reliably identified by the MALDI.

17 Primary MGIT Spectral Background MALDI analysis of primary MGIT cultures is impeded by background noise caused by culture components. To investigate and quantify this interference the following media conditions were extracted and analyzed in the absence of mycobacteria. MGIT Broth only MGIT +PANTA(10%) MGIT+sputum(6.25%) MGIT+PANTA(10%)/sputum(6.25%)

18 Primary MGIT Spectral Background BLUE= MGIT + PANTA RED= MGIT+ AFBnegative pooled sputa MGIT broth only 0 MGIT + PANTA 0 MGIT + AFBnegative pooled sputa MGIT + PANTA + AFB-negative pooled sputa Average MALDI Score

19 WSLH Algorithm for Primary Positive MGIT Tubes Specimen Triage Primary Positive MGIT tube Patient indicators TTD 5 days: TB/MAC PCR TTD < 5 days: Growth on BAP for 72 hours TB/MAC PCR MALDI-TOF Inconclusive results rpob/16s DNA Sequencing Good pure growth Report Conclusive Results 28

20 Evolution of WSLH ID Algorithm Culture containing AFB Previous HPLC Inconclusive results Accuprobes, Biochemicals DNA Sequencing Current TB/MAC PCR MALDI-TOF Inconclusive results DNA Sequencing 33

21 Questions?