What s Hot in Diagnostic Microbiology
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1 What s Hot in Diagnostic Microbiology Julianne Kus, MSc, PhD, FCCM Clinical Microbiologist Assistant Professor 1
2 Conflicts of Interest None No $ 2
3 Outline 1. Epidemiological Cut-off Values 2. Rapid Phenotypic Antimicrobial Susceptibility Testing 3. Hot Bugs 3
4 MaRS Discovery District 4
5 1. Epidemiological Cutoff Values J. Clin. Microbiol. May 2017 vol. 55 no
6 Epidemiological Cut-Off Values ECV/ECOFF = MIC value which identifies the upper limit of the wild-type (WT) population of a species to a drug Drug X / Bug Y > 3 labs > 100 isolates A microorganism is WT for the species by the absence of acquired and mutational mechanisms of resistance to the specific agent. ECV WT< mg/l Are generated with no clinical data Defined for specific bug-drug combinations in a defined phenotypic test system in vitro calculated statistically, estimated visually 6
7 ECVs vs Clinical Breakpoints ECV Clinical Breakpoint No fixed relationship Drug X / Bug Y 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 ECV WT< mg/l Clinical Breakpoint R> 0.25 mg/l Modified from: Turnidge & Paterson, Clin Micro Rev 2007;20:
8 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 M th ed. Appendix G 8
9 CLSI ECVs for Fungi Reference BMD Several Candida spp. Amphotericin B Fluconazole Itraconazole Posaconazole Voriconazole Micafungin Anidulifungin Cryptococcus Amphotericin B Fluconazole Itraconazole Posaconazole Voriconazole Flucytosine Several Aspergillus spp. Amphotericin B Voriconazole Itraconazole Posaconazole Isavuconazole Caspofungin CLIS M59-ED2: 2018 Lockhart et al JCM 55(5)
10 Azithromycin Shigella flexneri Shigella sonnei Neisseria gonorrhoeae Vancomycin CLSI ECVs for Bacteria Reference BMD (also via zone diameter) Cutibacterium acnes (aka Propionibacterium acnes) Clostridioides difficile Colistin Klebsiella aerogenes (aka Enterobacter aerogenes) Enterobacter cloacae E. coli Klebsiella pneumoniae Raoultella ornithinolytica (aka Clostridium difficile) CLIS M100: 2017, 2018 Appendix G 10
11 ! 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 12
13 2. Rapid Phenotypic Antimicrobial 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 (9): PLoS ONE (12):e
14 Image from: 14
15 Rapid Susceptibility Testing Blood drawn Blood culture incubation Gram Samples plated, incubate, ID Phenotypic Susceptibility Testing Routine Work Up t=0 t=8h t=20-36h t=38-72h Blood culture incubation Blood culture incubation Gram Direct MALDI t=0 t=8h Gram Molecular Panel t=0 t=8h Direct MALDI Syndromic Molecular Multiplex t=9-14h t=10-12h Large organism databases RUO select proteins involved in resistance: Bla KPC, PSM-mec, CfiA Select targets frequent BSI-causing bacteria/yeast CTX-M, bla KPC, bla NDM, bla VIM, bla IMP, bla OXA meca, vana/b proteins DNA J. Clin. Microbiol. September 2014 vol. 52 no PLoS ONE (12):e
16 Rapid Susceptibility Testing Blood drawn Blood culture incubation Gram Samples plated, incubate, ID Phenotypic Susceptibility Testing Routine Work Up t=0 t=8h t=20-36h t=38-72h Blood culture incubation Blood culture incubation Gram Direct MALDI t=0 t=8h Gram Molecular Panel t=0 t=8h Direct MALDI Syndromic Molecular Multiplex t=9-14h t=10-12h Select known markers of resistance Do not measure actual phenotypic response of the bug to the drug J. Clin. Microbiol. September 2014 vol. 52 no PLoS ONE (12):e
17 Rapid Phenotypic Susceptibility Testing Phenotypic Antimicrobial Susceptibility Tests (ASTs) directly assess if an antibiotic limits/stops microbial growth Technological and Methodological Approaches i. Beta-lactamase activity detection via MALDI ii. Single cell imaging and extrapolated MICs iii. Revised CLSI methods 17
18 Beta-lactamase activity detection via MALDI RUO software for MALDI ToF MS (MBT STAR-BL Bruker) Intact Beta- Lactam Hydrolysed Beta-Lactam Bacteria are co-incubated with a betalactam antibiotic. Beta-lactams hydrolysed in presence of appropriate beta-lactamase. Intact beta-lactam Hydrolysis results in a mass shift of the molecule (antibiotic) which can be measured using mass spectrometry. Hydrolysed beta-lactam Low molecular mass range ( Da) PLOS ONE April 6,
19 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 hrs 3. Investigate ß-lactamase activity via MALDI AMP, CTX, CAZ, PIP, MEM +BC (n=153) Gram stain MALDI Time=0 T > 5h ID & AST Lee AWT et al. 2018, 9(334):1-13 Routine culture, ID (MALDI), AST (disk diffusion) Potential reduction in TAT of hr T=52-58h ID & AST 19
20 Beta-lactamase activity detection via MALDI 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 Sensitivity * % Specificity * % Isolate Direct from BC Isolate Direct from BC Ampicillin Piperacillin Cannot detect non-hydrolytic betalactam resistance mechanisms, such as porin alterations and efflux mechanisms Limited to beta-lactam antibiotics Not an MIC 3rd gen Ceph Meropenem Lee AWT et al. Frontiers in Microbiology. 2018, 9(334):
21 1 2 AMP 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 DORI MECI AMOX-CLAV FOSFO NITRO CIPRO LEVO TMP/SMX PNAS (34):
22 April 2018 Vol 56 Issue 4 e Sept 2017 Vol89, Issue 1, Pages July 2017 Vol 55 Issue Feb 20. doi: /jac/dky032 April 2018 Vol 56 Issue 4 e Jan 2018 Vol 56 Issue 1 e
23 1 sample/unit 23
24 Specimen Prep Identification Susceptibility Gel-electro-filtration Electrokinetic concentration; FISH Morphokinetic Single Cell Analysis; MIC-based ASTs < 10 min ~1.5 hours ~7 hours 24
25 Specimen Prep Identification Susceptibility Morphokinetic Single Cell Analysis; MIC-based ASTs ~7 hours 25
26 April 2018 Vol 56 Issue 4 e academic medical centres Compared current Standard of Care (SOC) to Accelerate Pheno system for ID and AST of bacteria from positive Blood Cultures SOC : ID: gram stain, culture, MicroScan/MALDI +/- API AST: MicroScan WalkAway 96 Plus 26
27 FDA-cleared Bug-Drug Combinations Ampicillin Ceftaroline Erythromycin Daptomycin Linezolid Vancomycin MRSA MLSB S. aureus ü ü ü ü ü ü S. lugdunesis ü ü ü CNST ü ü ü ü E. faecium ü ü ü ü E. faecalis ü ü ü ü Streptococcus E.coli Klebsiella Enterobacter Proteus Citrobacter Amp-Sulbac Pip-Taz Cefipime Ceftazidime Ceftriaxone Ertapenem Meropenem Amikacin Gentamicin Tobramycin Ciprofloxicin Aztreonam ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü ü S. marcescens ü ü ü ü ü ü ü ü ü ü ü C. albicans C. glabrata P. aeruginosa ü ü ü ü ü ü ü ü A. baumanii ü ü Table 1. JCM 2018 Vol 56 Issue 4 e
28 Identification N=277 Sens (%) Spec (%) PPV (%) NPV (%) ALL Only organisms the system is approved to detect. #s are lower when ALL organisms identified during the study are included hours faster than current SOC 298 +BC AccelPheno 277 successful runs 218 definitive ID ID 218/298 =73.1% 28
29 Susceptibility CA (%) 298 +BC AccelPheno AST 146/218=70% GP (n= 242) GN (n=976) data points successful runs 218 definitive ID (on-panel) 146 available for AST analysis 59 GP 87 GN 40.6 hours faster than current SOC 29
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) 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 30
31 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) Best way to incorporate into lab/ utilization? Clinical outcome data needed Conclusions Overall provides accurate and rapid results for on panel organisms Currently cannot replace SOC, useful adjunct 31
32 Direct-from-Blood Culture AST testing 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 + - Disk diffusion - Automated Systems J. Clin. Microbiol. March :3 e
33 Direct-from-Blood Culture AST testing March :3 e Develop a standardized AST method direct from +BCs: Disk Diffusion à could be performed by all laboratories Simple to implement +
34 Towards development of a Revised CLSI method Reference broth microdilution AST GNB N = 20 Reference Disk Diffusion (CLSI) rdd 3 different commercial BC systems & bottles 1. Modify inoculum 2. Modify incubation time Save time drops 14 antibiotics 6 hour read 18 hour read 971 data points recorded Blood Agar Colony Counts MH Agar Direct Disk Diffusion ddd No standardization of cell density! 34
35 Modified Inoculum Findings and Conclusions 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
36 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
37 Rapid Phenotypic Susceptibility Testing Not 100% there yet but tools are being developed reduce TATs to help guide most appropriate therapy 37
38 38
39 3. Hot Bugs 39
40 Candida auris JCM T au ris. L. gen. f. n. auris, isolated from the ear discharge of a human patient Ovoid, ellipsoidal to elongate No pseudohyphae (?) Growth at 42 C, not 45 C 10um Candida haemulonii CBS5149 T Candida ruelliae CBS10815 T Candida heveicola CBS10701 T Candida pseudohaemulonii JCM12453 T Candida auris JCM15448 T ITS region Candida rugosa JCM1619 T Ø BSIs, elevated MICs to azoles, misidentified Microbiology and Immunology Volume 53, Issue 1, pages 41-44, 15 JAN 2009 DOI: /j x 40
41 Misidentified Highly transmissible between patients and environment MDR Nosocomial infections/outbreaks 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
42 Diagnostic Method Misidentified as: Comments API 20C Phoenix VITEK 2 YST MicroScan R. glutinis, C. sake, no ID C. haemulonii, C. catenulate, no ID C. haemulonii, C. lusitaniae, C. famata, C. duobushaemulonii, no ID C. lusitaniae, C. famata, C. guilliermondii, C. parapsilosis, C. albicans, C. tropicalis, no ID C. auris (8.01) MALDI ToF VITEK MS MALDI ToF Burker PCR & Sequencing ITS and D1-D2 C. haemulonii, no ID, C. pulcherrima, C. rugosa C. auris ( & RUO) C. auris (6903 or 7311) C. auris Jeffery-Smith A et al Candida auris: a review of the literature. Clin Microbiol Rev 31:e
43 The American experience thus far : 7 cases Current case count (as of March 31, 2018): confirmed cases colonized patients In the US: 90% resistant to fluconazole 30% resistant to amphotericin B 5% resistant to echinocandins U.S. hospitals identified C. auris in 5 patients who were recently hospitalized in other countries (India, Pakistan, South Africa, and Venezuela). 43
44 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 Jeffery-Smith A et al Clin Microbiol Rev 31:e
45 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
46 ITS and D1/D2 PCR & sequencing 30 PDA C 28 days 35 C um PDA 30 C 35 C 5um Emmonsia-like mycelia with florets 5um Yeast cells (2.5 5 um) Narrow-based buds 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): Dukik K, Muñoz JF, Jiang Y, et al. Novel taxa of thermally dimorphic systemic pathogens in the Ajellomycetaceae (Onygenales). Mycoses. 2017;60:
47 Emergomyces canadensis Recent global emergence of novel fungus Isolated from Canadian cases 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 My connect the dots 47
48 48
49 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 50
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