What’s Hot in Diagnostic Microbiology

Julianne Kus, MSc, PhD, FCCM

Clinical Microbiologist

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Assistant Professor

Conflicts of Interest• None• No $

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Outline1. Epidemiological Cut-off Values

2. Rapid Phenotypic Antimicrobial Susceptibility Testing

3. “Hot Bugs”

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MaRS Discovery Districthttps://marsdd.com/

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1. Epidemiological Cutoff Values

J. Clin. Microbiol. May 2017 vol. 55 no. 5 1262-1268 5

Epidemiological Cut-Off Values

• A microorganism is WT for the species by the absence of acquired and mutational mechanisms of resistance to the specific agent.

• Are generated with no clinical data

• Defined for specific bug-drug combinations in a defined phenotypic test system – in vitro

http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/EUCAST_Presentations/2011/EW1_Brown_Definitionsf2.pdf

ECVWT< 0.064 mg/L

> 3 labs> 100 isolates

• ECV/ECOFF = MIC value which identifies the upper limit of the wild-type (WT) population of a species to a drug

calculated statistically, estimated visually

Drug X / Bug Y

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ECVs vs Clinical Breakpoints

Clinical BreakpointR> 0.25 mg/L

ECVWT< 0.064 mg/L

Modified from: http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/EUCAST_Presentations/2011/EW1_Brown_Definitionsf2.pdf

Turnidge & Paterson, Clin Micro Rev 2007;20:391-408

Drug X / Bug Y

• ECV ≠ Clinical Breakpoint – No fixed relationship

• MIC > ECV microorganism likely to have an acquired form of resistance

• MIC < ECV microorganism is likely WT for a particular drug– does NOT mean Susceptible!! – could have Intrinsic resistance to that drug

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Why is CLSI providing ECVs?• When data is limited on:

– the correlation between MIC and clinical response (ideally from clinical trials)– in vivo and in vitro data– drug pharmacokinetics/pharmacodynamics

• ECVs easier to generate

• ECVs can be used to determine if an isolate is WT in regards to its in vitro response to an antimicrobial agent – do not predict of in vivo efficacy

CLSI M100 28th ed. Appendix G8

CLSI ECVs for FungiReference BMD

Several Candida spp.

• Amphotericin B• Fluconazole• Itraconazole• Posaconazole• Voriconazole• Micafungin• Anidulifungin

Several Aspergillus spp.

• Amphotericin B• Fluconazole• Itraconazole• Posaconazole• Voriconazole• Flucytosine

CLIS M59-ED2: 2018Lockhart et al. 2017. JCM 55(5)1262-1268

• Amphotericin B• Voriconazole• Itraconazole• Posaconazole• Isavuconazole• Caspofungin

Cryptococcus

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CLSI ECVs for BacteriaReference BMD

Azithromycin• Shigella flexneri (also via zone diameter)

• Shigella sonnei• Neisseria gonorrhoeae

Colistin• Klebsiella aerogenes

(aka Enterobacter aerogenes) • Enterobacter cloacae • E. coli• Klebsiella pneumoniae• Raoultella ornithinolytica

Vancomycin• Cutibacterium acnes

(aka Propionibacterium acnes) • Clostridioides difficile

(aka Clostridium difficile) CLIS M100: 2017, 2018Appendix G 10

How do we use them?• WT isolate does not guarantee a clinical efficacy of that

antimicrobial agent – But suggests no acquired/mutational resistance mechanism

BUT• A non-WT isolate suggests the isolate may not respond as

expected to an antimicrobial agent – may have an acquired/mutational resistance mechanism

• If a Clinical Breakpoint exists – use it!11

!

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2. Rapid PhenotypicAntimicrobial Susceptibility Testing (AST)• MALDI & syndromic molecular multiplex panels à faster results than

ever before

• Identification alone of bacteria directly from blood cultures (BCs) have demonstrated clear clinical benefits

• Help identify source of bacteremia

• Guide empiric therapy

• Identify when isolates are unlikely to be clinically significant

J. Clin. Microbiol. September 2014 52(9):3433-3436PLoS ONE. 2016 11(12):e0169332 13

Image from: http://www.medvet.umontreal.ca/rcrmb/dynamiques/Images/Notre%20recherche/Fairbrother_E%20coli.jpg 14

Blood culture incubation Samples plated, incubate, ID Phenotypic Susceptibility Testing

“Direct” MALDI

Gram

Blood culture incubation Gram

Blood culture incubation

Blood drawn

SyndromicMolecular Multiplex

t=0 t=38-72ht=20-36ht=8h

t=0 t=8h

t=0 t=8h

t=9-14h

t=10-12h

J. Clin. Microbiol. September 2014 vol. 52 no. 9 3433-3436PLoS ONE. 2016 11(12):e0169332https://www.luminexcorp.com/ http://www.biomerieux.com https://www.bruker.com

Rapid Susceptibility Testing

Gram

“Routine Work Up”

“Direct MALDI”

Molecular Panel

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Large organism databasesRUO – select proteins involved in resistance: BlaKPC, PSM-mec, CfiA

Select targets – frequent BSI-causing bacteria/yeast CTX-M, blaKPC, blaNDM, blaVIM, blaIMP, blaOXA

mecA, vanA/B

proteins

DNA

Blood culture incubation Samples plated, incubate, ID Phenotypic Susceptibility Testing

“Direct” MALDI

Gram

Blood culture incubation Gram

Blood culture incubation

Blood drawn

SyndromicMolecular Multiplex

t=0 t=38-72ht=20-36ht=8h

t=0 t=8h

t=0 t=8h

t=9-14h

t=10-12h

J. Clin. Microbiol. September 2014 vol. 52 no. 9 3433-3436PLoS ONE. 2016 11(12):e0169332https://www.luminexcorp.com/ http://www.biomerieux.com https://www.bruker.com

Rapid Susceptibility Testing

Gram

“Routine Work Up”

“Direct MALDI”

Molecular Panel

• Select known markers of resistance

• Do not measure actual phenotypic response of the bug to the drug

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Rapid Phenotypic Susceptibility Testing• Phenotypic Antimicrobial Susceptibility Tests (ASTs)

directly assess if an antibiotic limits/stops microbial growth

• Technological and Methodological Approachesi. Beta-lactamase activity detection via MALDIii. Single cell imaging and extrapolated MICsiii. Revised CLSI methods

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Beta-lactamase activity detection via MALDI

• RUO software for MALDI ToF MS (MBT STAR-BL Bruker)

• Bacteria are co-incubated with a beta-lactam antibiotic.

• Beta-lactams hydrolysed in presence of appropriate beta-lactamase.

• Hydrolysis results in a mass shift of the molecule (antibiotic) which can be measured using mass spectrometry.

• Low molecular mass range (100 – 1000 Da)

Hydrolysed Beta-Lactam

Intact Beta-Lactam

PLOS ONE | https://doi.org/10.1371/journal.pone.0174908 April 6, 2017

Intact beta-lactam

Hydrolysed beta-lactam

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Beta-lactamase activity detection via MALDI• 200 retrospective, 153 prospective

Blood cultures with GNBs• Variety of known resistance

mechanisms• Compared to routine ID and AST

1. Direct ID by MALDI from blood (saponin-based extraction)

2. Incubate bacteria + ß-lactam – 2 hrs3. Investigate ß-lactamase activity via

MALDI

• AMP, CTX, CAZ, PIP, MEMPotential reduction in TAT of

47 - 53 hrTime=0

T > 5hID & AST

T=52-58h ID & AST

+BC (n=153)

Gram stain Routine culture, ID (MALDI), AST (disk diffusion)

MALDI

Lee AWT et al. 2018, 9(334):1-13

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Beta-lactamase activity detection via MALDI

Lee AWT et al. Frontiers in Microbiology. 2018, 9(334):1-13

Sensitivity* % Specificity* %

Isolate Direct from BC

Isolate Direct from BC

Ampicillin 91.3 80.4 100 92.9

Piperacillin 100 100 100 100

3rd gen Ceph 97.9 68.8 100 91.5

Meropenem 100 40 100 97.7

þ Detection of ß-lactamase activity from polymicrobial cultures (14/19)

ý Rapid but still issues with sensitivity direct from BCs– False Negatives and False Positives

• Cannot detect non-hydrolytic beta-lactam resistance mechanisms, such as porin alterations and efflux mechanisms

• Limited to beta-lactam antibiotics

• Not an MIC

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PNAS 2017 114(34): 9170–9175www.pnas.org/cgi/doi/10.1073/pnas.1708558114

• Microfluidics and direct single-cell imaging

• Detection of response to antibiotic treatment through monitoring of cell growth rate

• detection of response of susceptible E. coli cells 3-10 minutes

AMP AMOX-CLAV CIPRO

DORI FOSFO LEVO

MECI NITRO TMP/SMX

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1

2

April 2018 Vol 56 Issue 4 e01329-17

April 2018 Vol 56 Issue 4 e01672-17

2018 Feb 20. doi: 10.1093/jac/dky032

Jan 2018 Vol 56 Issue 1 e01166-17

July 2017 Vol 55 Issue 7

Sept 2017 Vol89, Issue 1, Pages 52–57

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1 sample/unit

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Specimen Prep

Identification

Susceptibility

Gel-electro-filtration

Electrokineticconcentration; FISH

Morphokinetic Single Cell Analysis;

MIC-based ASTs

http://acceleratediagnostics.com/technology/mca/

< 10 min

~1.5 hours

~7 hours

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Specimen Prep

Identification

Susceptibility Morphokinetic Single Cell Analysis;

MIC-based ASTs25

~7 hours

• 2 academic medical centres

• Compared current Standard of Care (SOC) to Accelerate Phenosystem for ID and AST of bacteria from positive Blood Cultures

• SOC :– ID: gram stain, culture, MicroScan/MALDI +/- API

– AST: MicroScan WalkAway 96 Plus

April 2018 Vol 56 Issue 4 e01672-17

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FDA-cleared Bug-Drug Combinations

Amp-

Sulb

ac

Pip-

Taz

Cefip

ime

Cefta

zidim

e

Ceftr

iaxo

ne

Erta

pene

m

Mer

open

em

Amik

acin

Gent

amici

n

Tobr

amyc

in

Cipr

oflo

xicin

Aztre

onam

E.coli ü ü ü ü ü ü ü ü ü ü ü üKlebsiella ü ü ü ü ü ü ü ü ü ü ü üEnterobacter ü ü ü ü ü ü ü ü ü ü üProteus ü ü ü ü ü ü ü ü ü ü ü üCitrobacter ü ü ü ü ü ü ü ü ü ü üS. marcescens ü ü ü ü ü ü ü ü ü ü üP. aeruginosa ü ü ü ü ü ü ü üA. baumanii ü ü

Ampi

cillin

Cefta

rolin

e

Eryt

hrom

ycin

Dapt

omyc

in

Linez

olid

Vanc

omyc

in

MRS

A

MLS

B

S. aureus ü ü ü ü ü üS. lugdunesis ü ü üCNST ü ü ü üE. faecium ü ü ü üE. faecalis ü ü ü üStreptococcus

C. albicans

C. glabrata

Table 1. JCM 2018 Vol 56 Issue 4 e01672-1727

IdentificationN=277 Sens

(%)Spec (%)

PPV (%)

NPV (%)

ALL 94.7 98.9 83.7 99.7

Only organisms the system is approved to detect. #s are lower when ALL organisms identified during the study are included.

39.9 hours faster than current SOC

ID218/298 =73.1%

298 +BCAccelPheno

277 successful

runs

218 definitive

ID

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298 +BCAccelPheno

277 successful

runs

218 definitive

ID(on-panel)

146 available for AST analysis

59 GP87 GN

40.6 hours faster than current SOC

SusceptibilityCA (%)

GP (n= 242) 97.1

GN(n=976) 93.3

data points

AST146/218=70%

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• At least 26.8% of the time routine ID and AST still required– Technical failures (n=21); Indeterminate (n=36); Off-panel organisms (n=23)

• False-positives (n=39)– eg. 11 false-positive S. aureus à 8 were actually CNST

• False-negatives (n=10)

Conclusions• Overall provides accurate and rapid results for on panel

organisms• Currently cannot fully replace SOC, useful adjunct

Caveats & Conclusions

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Caveats & Conclusions• At least 26.8% of the time routine ID and AST still required

– Technical failures (n=21); Indeterminate (n=36); Off-panel organisms (n=23)

• Significant # of false-positives (n=39)– 11 false-positive S. aureus à 8 were actually CNST ; 8 C. glabrata

• False-negatives (n=10)

Conclusions• Overall provides accurate and rapid results for on panel

organisms• Currently cannot replace SOC, useful adjunct

• Best way to incorporate into lab/ utilization?

• Clinical outcome data needed

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Direct-from-Blood Culture AST testing

J. Clin. Microbiol. March 2018 56:3 e01678-17

+ - Disk diffusion- Automated

Systems

• Try to detect resistance faster – 1 day earlier

• CLSI conducted a survey about this practice:– Most labs reported excellent performance

– Results often not officially reported

– No standardization of methods between laboratories

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Direct-from-Blood Culture AST testing

+• Develop a standardized AST method direct from +BCs:– Disk Diffusion à could be performed by all laboratories– Simple to implement

March 2018 56:3 e01678-17

Towards development of a Revised CLSI method

1. Modify inoculum2. Modify incubation time

• 971 data points recorded

GNB N = 20

Reference broth

microdilutionAST

Reference Disk Diffusion (CLSI)

rDD

+.

.

.

... .

..

.

. .

Blood AgarColony Counts

MH AgarDirect Disk Diffusion

dDD

6 hour read18 hour read

3 different commercial BC systems & bottles

14 antibiotics

4 drops

No standardization of cell density!

Save time

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Findings and ConclusionsModified Inoculum

• Overall Categorical Agreement at 18 hours between rDD and dDD = 88.7%

• Challenge:àConcentration of bacteria between the different commercial BC bottles after incubation differed significantly (spanning 3 logs)

à led to variable amounts of bacteria plated

à Would need to be standardized

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Modified Incubation Time• 6 hour incubation – too difficult to read - insufficient growth

• Overall Categorical Agreement of dDD at 6 h with rDD at 18 h = 70%(of those that were able to be evaluated (n=772))

• Discrepancies between 6 and 18 hour times points– Insufficient growth, or incomplete diffusion of antibiotic into agar– CLSI is investigating an 8-10 hour “early read” point

• Will form basis of a larger multicentre study

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• Not 100% there yet but tools are being developed reduce TATs to help guide most appropriate therapy

Rapid Phenotypic Susceptibility Testing

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3. “Hot” Bugs

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Candida auris

Ø BSIs, elevated MICs to azoles, misidentified40

au’ris. L. gen. f. n. auris, isolated from the ear discharge of a human patient

Microbiology and Immunology Volume 53, Issue 1, pages 41-44, 15 JAN 2009 DOI: 10.1111/j.1348-0421.2008.00083.x

JCM 15448T

Ovoid, ellipsoidal to elongateNo pseudohyphae (?)Growth at 42◦C, not 45◦C

ITS region

Candida haemulonii CBS5149T

Candida pseudohaemulonii JCM12453T

Candida ruelliae CBS10815T

Candida heveicola CBS10701T

Candida auris JCM15448T

Candida rugosa JCM1619T

10um

• Misidentified• Highly transmissible between

patients and environment– Nosocomial infections/outbreaks

• MDR• Associated with high mortality• Globally widespread

Isolated from a range of body sites• skin (very common)• urogenital tract (common)• respiratory tract (occasional)• invasive infections

– candidaemia– pericarditis– urinary tract infections– pneumonia

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Diagnostic Method Misidentified as: Comments

API 20C R. glutinis, C. sake, no ID

Phoenix C. haemulonii, C. catenulate, no ID

VITEK 2 YST C. haemulonii, C. lusitaniae, C. famata, C. duobushaemulonii, no ID

C. auris(8.01)

MicroScan C. lusitaniae, C. famata, C. guilliermondii, C. parapsilosis, C. albicans, C. tropicalis, no ID

MALDI ToF – VITEK MS

C. haemulonii, no ID,C. pulcherrima, C. rugosa

C. auris(4.4.0-16 & RUO)

MALDI ToF – Burker C. auris(6903 or 7311)

PCR & Sequencing ITS and D1-D2

C. auris

https://www.phe-culturecollections.org.uk/news/ncpf-news/ncpf-in-research-pathogenicity-of-candida-auris-an-emerging-pathogen.aspxJeffery-Smith A et al. 2018. Candida auris: a review of the literature. Clin Microbiol Rev 31:e00029-17

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The American experience thus far 2013 -2016: 7 cases

Current case count(as of March 31, 2018):

- 257 confirmed cases- 475 colonized patients

https://www.cdc.gov/fungal/diseases/candidiasis/tracking-c-auris.html

U.S. hospitals identified C. auris in 5 patients who were recently hospitalized in other countries(India, Pakistan, South Africa, and Venezuela).

In the US: 90% resistant to fluconazole30% resistant to amphotericin B5% resistant to echinocandins

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Jeffery-Smith A et al. 2018. Clin Microbiol Rev 31:e00029-17

• Previous hospitalization & procedure in India

• Brain abscess & chronic otitis externa

• Isolate form ear fluid C. auris• Resistant to FLU and Ampho B• Pt also had a CRO/CPE• No know transmission

What is happening in Canada?• A few cases of C. auris identified in Canada – no known outbreaks (as of

date of preparation)

• CNISP– “C. auris interest group”

– Includes CNISP members as well other representatives (NML and PHO)

– Determine preparedness amongst Canadian labs and IPAC teams regarding screening and testing

• CPHLN/PHAC/NML– Development of the “NML Mycology Reference Centre”

• CPHLN Provincial Mycology Leads providing expert advice

• Plan to provide C. auris identification confirmation +/- susceptibility testing, WGS outbreak support

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Yeast cells (2.5 – 5 um)Narrow-based buds

Emmonsia-like mycelia with florets

PDA 28 days

5um5um Schwartz IS, Sanche S, Wiederhold NP, Patterson TF, Sigler L. Emergomyces canadensis, a Dimorphic Fungus Causing Fatal Systemic Human Disease in North America. Emerg Infect Dis. 2018;24(4):758-761. https://dx.doi.org/10.3201/eid2404.171765

Dukik K, Muñoz JF, Jiang Y, et al. Novel taxa of thermally dimorphic systemic pathogens in theAjellomycetaceae (Onygenales). Mycoses. 2017;60:296–309.https://doi.org/10.1111/myc.12601

ITS and D1/D2 PCR & sequencing

2003

199230◦C 35◦C

10um

PDA30◦C 35◦C

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“Emergomyces canadensis”

• Newly described North American species, of the relatively newly described genus of pathogenic dimorphic fungus

• 4 known cases

• Ajellomycetaceae (same as Blastomyces and Histoplasma)

• Members of the genus known to cause disseminated disease in immunocompromised patients – Es. africanus

Recent global emergenceof novel fungus

Isolated from Canadian cases

My “connect the dots”

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http://don.komarechka.com/2012/04/fisheye-toronto/48

The Wrap-Up• Clinical break points for bug-drug combinations are

difficult to develop; ECVs will likely be increasingly available to help guide therapeutic decisions

• New technologies and methodological changes will soon allow for quicker reporting of phenotypic susceptibility results

• Don’t forget the fungi!49

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