9/2/2014

Effective Communication?  How to better pose the ?

MALDI-TOF and CF Microbiology Robert C Jerris, PhD, D(ABMM) Medical Director, Clinical Microbiology Children’s Healthcare of Atlanta Emory University School of Medicine 1

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Well you told me not to write on the walls!!

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Objectives Children’s by the Numbers 3 Hospitals 520 staffed beds 17 neighborhood locations, including: Four Immediate Care Centers One Primary Care Center Marcus Autism Center More than 7,500 employees Access to more than 1,600 pediatric physicians 6,500 volunteers 2nd Largest population of pediatric CF patients in US

 CF Basics • CF foundation info • Impact of microbiome and population microbiology  Through the EM and microgenomics  Through a looking glass  Procedures • From specimen type-to plating and examination of organisms-to susceptibility testing  Our experience with MALDI-TOF

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9/2/2014

CF microbiome through the EM: Expanded Profiling of CF Airway Microbiology

Contemporary Issues in CF Microbiology  Increased Life Expectancy  Development and use of broad spectrum antimicrobials • Shift in microbiome to more resistant organisms with questionable or low virulence  Diagnostic advances esp. sequence analysis (entire microbiome) and MALDI-TOF (cultivable microbiome)

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Microbiology: through the looking Glass Standard Culture-Dependent Profiling

Sibley CD, Grinwis ME, Field TR, Eshaghurshan CS, Faria MM, et al. (2011) Culture Enriched Molecular Profiling of the Cystic Fibrosis Airway Microbiome. PLoS ONE 6(7): e22702. doi:10.1371/journal.pone.0022702

Impact of MALDI  Will allow for EXHAUSTIVE *MICRO from cultivable organisms  AEROBES  ANAEROBES  MICROAEROPHILIC  MICROAEROBIC  FUNGI (yeast)

Bacterial genera recovered from CF sputum during 28 years of using conventional cultivation approaches, 19,250 isolates. (Southern Alberta Cystic Fibrosis Clinic)

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Sibley CD, Grinwis ME, Field TR, Eshaghurshan CS, Faria MM, et al. (2011) Culture Enriched Molecular Profiling of the Cystic Fibrosis Airway Microbiome. PLoS ONE 6(7): e22702. doi:10.1371/journal.pone.0022702

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*, database dependent

CUCYST: Bacterial Culture, Cystic Fibrosis, Respiratory Routine: Gram Stain: BAL only, no rejection criteria Media: 35-37oC, minimum 4 days Chocolate agar +/- bacitracin (5% CO2) Sheep blood agar (5% CO2) MacConkey agar BCSA agar Staphylococus selective/differential MSA agar, Columbia colistin-nalidixic acid agar (CNA) [also good for pneumococcus,NTM], Chromagar

Frequency (non-transplant): Annual and Quarterly Visits, Exacerbations; no more than 1/mo routine with referral to previous culture as appropriate

 Specimen type: • Sputum (expectorated or induced) [sterile container] • Throat swab (gagged) [Stuart’s Transport] • Bronchial aspirate or washing, brochoalveolar lavage [sterile container][if quant ≥103] • Endotracheal aspirates (ventilated pt)

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 Transport delay: Refrigerate = 1.7 : Genus >=2.0 : Genus ,sp.

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Direct Extraction:  Place organism directly onto template (we use toothpicks)  Add1 ųl freshly prepared 70% formic acid  Allow to dry  Add 1 ųl matrix ; dry  MALDI analysis

Standard Ethanol/Formic Acid Extraction:

2 spots per sample (increases sensitivity to >95%) Air dry; add 1 µl Matrix (α-cyano-4-hydroxycinnamic acid); air dry Analyze in instrument

 Suspended organisms in 300 ųl sterile water (we use NA free from molecular lab)  Add 900 ųl of 100% Ethanol  Vortex to mix thoroughly  Centrifuge 13,000rpm (20,000 xg) for 2 minutes.  Remove supernatant  Repeat  Leave cap open for ~10 min to dry pellet (or dry under nitrogen or Speedy Vac )  Add 50 ųl of freshly prepared 70% formic acid ; mix thoroughly  Add 50 ųl 100% acetonitrile  Vortex to mix thoroughly  Centrifuge 13,000rpm (20,000 xg) for 2 minutes.  Add 1 ųl of supernatant to target  Allow to dry  Add1 ųl matrix  MALDI analysis

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9/2/2014

Discrepant Conventional ID vs. MALDI and Reference (N=32)

CF BCC/NFR Study Reference Sequence Analysis

 CHOA: Cepacia Ref Lab: Jerris-LiPuma collaborative • Double Blind Study  API NE and MicroScan (Prompt and Dilution methods) vs MALDI/Ref Lab

Achomobacter xyl 00066376

Burkolderia multivorans

B. multivorans

Pandoraea pnomenusa

P. apista (1.9)

A. xylo 04066376

B.gladioli

B.gladioli

Ps. stutzeri 00060662

BioMerieux API NE

B cepacia 99.9% Low Probability (1067577) 48h; LOW disc, Alc.faecalis 67.6% Ach. xylo 40067376 (1000457)

B. cenocepacia

B.cenocepacia

B. cepacia 00041774

Nonfermenter sp./Low probability B. cepacia 05041776

B.cenocepacia

B.cenocepacia

B. cepacia 04040776

Nonfermenter sp./Low probability

S. maltophilia

S. maltophilia

Ach. xylo 04062736

B. cepacia 04062776

S. maltophilia 99.9% (1472345)

 MALDI vs recA sequence analysis (B cepacia

Reference Lab)

B.cenocepacia 20

B cepacia 99.9% (1067573) 48h; No Valid ID (1050552) 48h; UNACCEPT (1057500)

B.multivorans

Ach. xylo 00067776

Nonfermenter sp.

48h; B.cepacia 99.9% (1067577)

B.cenocepacia

Ralstonia pick. 00060756

Low prob R pickettii(65%)

48h, UNACCEPT (1050540)

B. cepacia 04041772

B. cepacia 04061772

48h; UNACCEPT (0472550)

B.multivorans

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MicroScan Prompt Broth

MALDI

B.cepacia

B.cepacia

Table 1:  Organisms Commonly Isolated From CF Patients:  Comparison of Conventional or Reference Methods* to MALDI‐TOF MS

Conventional vs MALDI/Ref for ID

(Desai AP, Stanley T, Atuan M, et al. J Clin Pathol (2012). doi:10.1136/jclinpath-2012-200772)

No. of Isolates

• For 32 NFR CF isolates, agreement with reference ID was 81%, 78% and 85%, respectively for MSP, MSB, and API NE.

Organism Non‐fermenters

• BCC (N=29) was misidentified as other organisms for 5 isolates by MSP, 2 for MSB and 0 for API NE (‘unacceptable ID’). One strain of S. maltophilia was id’d as BCC by MSB.

(% of total  tested)

No. (%) of Isolates Agreement to  Genus, species  Level

Agreement to  Genus Only

12 (57)

Achromobacter xylosoxidans 

21 (5)

9 (43)

Acinetobacter baumannii

11 (2)

8 (73)

Acinetobacter lwoffii

3 (