Research Opportunities in Clinical Microbiology

Kimberly Hanson, MD, MHS Associate Professor of Medicine and Pathology

Section Head, Clinical Mycology Laboratory Medical Director, Transplant Infectious Diseases

Program Director, Medical Microbiology Fellowship

University of Utah and ARUP Laboratories

Medical Microbiology

Overview • Subspecialty Pathology

– 1 year training program after • Adult or Pediatric ID • Anatomic/Clinical Pathology

– Certification • Direct a laboratory • Enhance ID skills/knowledge • Opportunities for research

– Organism specific – Drug Resistance – Diagnostics – Outcomes research – Epidemiology – And the list goes on……….

ACGME Accredited Programs 1. Beth Israel Deaconess 2. Brigham & Women’s Hospital 3. Cleveland Clinic 4. Duke University 5. Emory University 6. Indiana University 7. John’s Hopkins University 8. Massachusetts General Hospital 9. Mayo Clinic 10. University of Chicago/North Shore 11. University of Iowa 12. University Texas Southwestern 13. University of Utah 14. Yale University

http://www.acgme.org/ads/Public/Programs/Search? Accessed June 2105

Research Focus Development and Validation of New Diagnostic Tests

Early phase test development Later phase test utilization

Invention Utilization

Several examples of recent/ongoing projects - Rapid identification of mold - Inducible macrolide resistance in RGM

Classic Fungal Identification Tests Method Advantages Disadvantages Culture • Isolate for ID and

susceptibility testing • Delayed results • Poor sensitivity • Lacks specificity from non-

sterile sites (colonization vs. infection vs. contamination)

Phenotypic ID • Most common fungi can be identified accurately

• Tease prep is fast and cheap

• Molds require expertise • Biochemical incubation 24-

72hrs • Misidentifications

Histopathology • Gold standard for invasive disease

• Difficult to obtain • Not species-specific • Prone to sampling bias

Nuclear rRNA 18S 5.8S Nuclear rRNA 28S ITS I ITS II

PCR

Sequencing

Fungal Sequence Database

Ribosomal RNA Gene Cluster

Gene Targets for Fungal Identification

Highly conserved Highly conserved

D1/D2 50-100 copies

Haploid genome

Current diagnostic standard for ID is expensive and technically complex

Approximately 2% of yeast go unidentified with combo phenotypic and genotypic testing Slechta ES, Hohmann SL, Simmon K, Hanson KE. Med Mycol. 2012 50(5):458-66

Complexities associated with Mold Identification Paecilomyces spp. are morphologically similar, but phyolgenetically diverse

2. Barker AP, Horan JL, Slechta ES, Alexander BD, and Hanson KE. Med Mycol 2013 ;52(5):53 1. JCM 2010; 48(8): 2754

Morphology Clinical Isolates (n=77) • Phenotypic ID

– P. lilacinus (n= 52) – P. variotii (n=21) – Paecilomyces spp. (n=4)

• Multi-locus sequencing (MLS) – ITS 1/2, D1/D2, β-tubulin – P. variotti phenotype or Paecilomyces spp.

• P. variotii sensu stricto (n=12) • P. formosus (n=3) • Paecilomyces dactylethromorphus (n=3) • Rasamsonia argillacea (n=4) • Rasamsonia piperina (n=1) • Hamigera spp. (n=1)

Required MLS

Matrix Assisted Laser Desorption Ionization (MALDI) Time-of-Flight (TOF) Mass Spectrometry (MS)

Organism Score

C. glabrata 2.1

C. albicans 1.8

1.) Pure Colony 2.) Protein Extraction a. Colonies mixed with 70% ETOH b. Centrifuge 20,800g x 2min c. Decant and dry pellet d. 70% formic acid and vortex e. Acetonitrile and vortex f. Centrifuge 20,800g x 2min g. 1μL supernatant onto target; dry h. 2 μL matrix; dry

5.) Interpretation • ≥ 2.0 and Δ10% species • 1.7-1.9 genus • < 1.7 no ID

3.)

4.) Spectra

Complexities associated with Mold Identification Is MALDI-TOF MS an easier and equally accurate option?

peak intensities represented in arbitrary units m/z = mass over charge ratio

MALDI-TOF MS Type Strain Spectra Principal Component Analysis-based Clustering

A. Purpureocillium lilacinum B. Paecilomyces dactylethromorphus C. Paecilomyces species D. Hamigera species E. Rasamsonia argillacea/piperina F. Paecilomyces variotii G. Paecilomyces formosus H. *could not be identified using the supplemented MALDI-TOF MS database.

Spectra derived from the Clinical Study Isolates

Barker AP, Horan JL, Slechta ES, Alexander BD, and Hanson KE. Med Mycol 2013 ;52(5):53

93.5% (72/77) agreement between the molecular and proteomic methods only after supplementation of the MALDI-TOF MS database with type strains

MALDI-TOF MS for Molds • Preliminary Data

– Custom spectral database: 162 MSPs, 41 genera, 61 species – Optimized extraction: glass beads with brief vortex – Prospective evaluation of 149 clinical isolates

ID Method Number of IDs Median Time to ID Comment

Phenotypic 105/149 (70%) 5 days Genus or complex-level ID only

Genotypic 34/44 (77%) 7 days Only performed when no phenotypic ID

MALDI 94/149 (63%)

2 days Single attempt with automatic fire 11 with no peaks

Slechta ES, Barker A, Powers-Fletcher M and Hanson KE. ASM Abstract; New Orleans, LA 2015

Phenotype and Maldi

Agree 74%

No MALDI ID

23%

No MALDI Peaks

3%

Phenotypic IDs N=105 No Phenotypic

ID and No MALDI ID

74%

MALDI ID 26%

No Phenotypic ID N=44

All black molds Not well represented in database

Mostly basidiomycetes

Non-sporulating or dermatophytes

Pan-fungal PCR Approaches • A “broad-range” of fungi can cause invasive disease

• Applied directly to clinical specimens or cultured isolates

– Tissue is of particular interest because specimens for culture are not always sent or remain negative even when hyphal elements are seen histopathologically

• A variety of amplicon identification methods exit:

– Examples: DNA sequencing, PCR-electrospray ionization mass spectroscopy, microarray

Hyaline Mold Histopathology (40x) Scedosporium Fusarium Scopulariopsis

Of 122 Non-pigmented (hyaline) septated hyphae with acute angle branching, 83% were confirmed Aspergillus spp. (Lee et al. Med Mycol; 2010: 48: 886)

Antifungal Activity Profile of Various Agents

Organism

Antifungal Drug Amphotericin

Voriconazole

Posaconazole

Echinocandins

Fusarium - ± ± - Scedosporium

apiospermum - + + ±

prolificans - - - - Paecilomyces lilacinus - + + - variottii + - + + Scopulariopsis - ± ± - Mucorales + - ± -

Assay variables affecting analytical sensitivity Primer Design

• 50 rRNA operon sequences – 27 broad-range primers

• Amplicons selected for length and human DNA amplification

• 11 amplicons screened for analytic sensitivity

– 30 fg DNA – 26 fungi in 14 genera

• 2 primer pairs selected – ITS2 (5.8S-1R) detected all fungi – 28S (10F-12R) strongest bands

Tissue Extraction Method

Specimen Extraction Median Cts

Spiked tissue • 3 molds • 3 yeasts • 3 replicates

Chemagic Failed

Chemagic + Beads Failed

Maxwell 20

Maxwell + Beads 20.8

MoBio+ Beads 20.8

Zymo + Beads 20.2

Tissue only • 3 replicates

Chemagic Failed

Chemagic + Beads Failed

Maxwell 37.1

Maxwell + Beads 36.5

MoBio + Beads 42.0

Zymo + Beads 34.8 Khot et al. Appl. Environ. Microbiol. 2009;75:1559

Fungal DNA Contamination • Phlebotomy tubes1

– 18% had Aspergillus spp. DNA

• Reagents2 – Zymolase, lyticase, proteinase K

1.) Harrison et al. Diagn Microbiol Infect Dis 2010; 67: 392-394; 2.) Loeffler et al. J Clin Microbiol 1999; 37: 1200-1202;

30

35

40

45

0 2 4 6 8

Number of no template controls

Commercial “Fungal” Master Mix with Broad-range PCR

PCR

Ct

18S Primers 28S Primers ITS-2 Primers

30

35

40

45

0 5 10 1530

35

40

45

0 5 10 15 20

Graphs courtesy P. Khot PhD

• Mitigation Strategies Filter UV Light Environmental cleaning New blades Duplicate testing

Rapid Detection of Macrolide Resistance

M. abscessus group erm(41) gene

• Mycobacterium chelonae-abscessus complex – Acquired and inducible resistance mechanisms

• 23S rRNA peptidyltransferase gene (rrl) – Detected by standard 3 day susceptibility testing

• Erythromycin ribosomal methylase gene (erm) – Detected by extended incubation in clarithromycin (CLSI 2010)

» M. chelonae clarithromycin susceptible » M. abscessus group erm41

• M. abscessus subp abscessus (C to T position 28 (T28) resistant; C28 susceptible) • M. abscessus subp bolletti (T28) • M. abscessus subp. massiliense (247 bp deletion susceptible)

3 days 5 days 7 days 12 days 14 daysM. chelonae % (n=427) 100 100 100 100 99.8M. abscessus group % (n=1025) 97.3 90 63.1 48.6 37.9

0

20

40

60

80

100

% Is

olat

es S

usce

ptib

le

One M. chelonae and 3 M. abscessus group isolates had intermediate clarithromycin susceptibility (MIC 4 µg/ml) detected at day 14 on repeated testing. These organisms are classified as non-susceptible in the figure.

14 day clarithromycin susceptibility test results for all isolates over a 2 year period

Hanson KE, Slechta ES, Muir H, Barker AP.J Clin Microbiol. 2014 May;52(5):1705-7.

ITS Identification

14 Day Clarithromycin Susceptibility*

No. (%)

erm41 genotype

Susceptible

MIC ≤ 2 µg/ml

Intermediate

MIC 4 µg/ml

Resistant No.

MIC ≥ 8 µg/ml

M. Chelonae (n=45) 44 (97.8) 1 (2.2) 0 Not present

M. abscessus group (n= 285) 74 (26) 2 (0.7) 0 C28 sequevar

0 94 (33.0) T28 sequevar

114 (40) 1 (0.3) 0 Deletions

Correlation between erm(41) genotype and macrolide susceptibility

• All sequenced isolates were clarithromycin susceptible after 72 hours incubation in an attempt to eliminate organisms with acquired macrolide resistance due to rrl mutation.

Hanson KE, Slechta ES, Muir H, Barker AP. J Clin Microbiol. 2014 May;52(5):1705-7

So what came out of this? - multiplex PCR for simultaneous detection of rrl and erm 41 - recommendations for CLSI - f/u work with Tyler Texas – absence of inducible resistance in M. mucogenicum and M. immunogenum -

Brown-Elliott BA, Hanson KE et al. J Clin Microbiol. 2015 Mar;53(3):875-8

The Mycology R&D Team Medical technicians, Microbiology and ID fellows, Research Scientists, Junior Faculty

• Sue Slechta, PhD • Maggie Powers, PhD • Prasanna Khot, PhD • Erin Graff, PhD • Keith Simmon, PhD • Tatum Lunt, MS • Adam Barker, PhD

Work – Life Balance

University of Utah view from Dr. Sankar Swaminathan’s office