What’s Hot in Diagnostic Microbiology
Julianne Kus, MSc, PhD, FCCM
Clinical Microbiologist
1
Assistant Professor
Conflicts of Interest• None• No $
2
Outline1. Epidemiological Cut-off Values
2. Rapid Phenotypic Antimicrobial Susceptibility Testing
3. “Hot Bugs”
3
MaRS Discovery Districthttps://marsdd.com/
4
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
6
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
7
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
9
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
!
12
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
15
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
16
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
17
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
18
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
19
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
20
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
21
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
22
1 sample/unit
23
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
24
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
26
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
28
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%
29
• 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
30
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
31
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
32
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
34
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
35
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
36
• Not 100% there yet but tools are being developed reduce TATs to help guide most appropriate therapy
Rapid Phenotypic Susceptibility Testing
37
38
3. “Hot” Bugs
39
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
41
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
42
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
43
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
45
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
46
“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”
47
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
50