Research what you see: Antimicrobial resistance and pathogenesis

Research what you see: Antimicrobial resistance and pathogenesis Director, Mycology Research Unit and XDR Pathogen Laboratory University of Pittsburgh Cornelius J. Clancy, M.D. Chief, Infectious Diseases VA Pittsburgh Healthcare System NIAID/IDSA Infectious Diseases Research Career Meeting Bethesda, MD June 5, 2015

3 Allow myself to introduce myself

4 Allow myself to introduce myself

Candida infections Oropharyngeal candidiasis Candidemia Asmundsdottir J Clin MIcrobiol 2002 5 Rex NEJM 1994

What kinds of candidiasis are we seeing? Case reports Case series Clin Infect Dis 1997 6

What kinds of candidiasis are we seeing? Case reports Case series Clin Infect Dis 1997 Why do patients fail antifungal treatment? Clinical studies In vitro susceptibility testing AAC 1999 7

What kinds of candidiasis are we seeing? Case reports Case series Clin Infect Dis 1997 Why do patients fail antifungal treatment? Clinical studies In vitro susceptibility testing AAC 1999 How do Candida become resistant? How do they cause different infections? 8 Molecular biology Animal models DC, OPC, VVC Infect Immun 2003

IDSA Fellowship in Medical Mycology NIH KO8 Career Development Award (VA Career Development Award) 9

A case Case from 1 UPMC A 64 year-old man underwent right extended hepatectomy with roux en y biliary reconstruction for a non-malignant hepatic mass His post-operative course was complicated by biliary leak abscess s/p stent and ex-lap drainage, which revealed E. coli and VRE He underwent repeat laparotomies at 4 and 8 weeks after the hepatectomy

A case Case from 1 UPMC Abscess cultures at both times revealed C. glabrata and VRE Blood cultures negative He received 2 x 4 week courses of micafungin He developed septic shock Blood culture positive for C. glabrata He was treated with LFAB, but died

What is likelihood that this C. glabrata is echinocandin resistant? a. 0-5% b. 8-18% c. ~50% d. ~80% e. Almost 100% A case from UPMC

Echinocandins Inhibit β-1,3-glucan synthase Fungicidal in vitro; prolonged PAFE Induce Candida apoptosis Promote phagocytosis via Dectin-1 Highly active against C. albicans, C. glabrata, C. tropicalis, C. krusei Potential hole: C. parapsilosis ( MICs, significance?) Parenteral only Linear PK; amenable to dose-escalation Extremely high protein binding Penetration limited into urine, CNS, ocular fluid Excellent safety profile

Echinocandin resistance Garcia-Effron AAC 2008

Candida FKS mutant Candida Pittsburgh (2007-2014) 8% FKS mutant C. glabrata Shields AAC 2012, 2013; Shields 2015 Houston (2009-2012) 18% FKS mutant C. glabrata Beyda CID 2014 Duke (2001-2010) 8% FKS mutant C. glabrata Alexander CID 2013 FKS mutations are rare among other spp. C. albicans 1-5% Castanheira AAC 2010; Shields ICAAC 2014, 2015 C. tropicalis, C. krusei Castanheira AAC 2010; Prigitano New Microbiol 2014; Jensen AAC 2014; Jensen AAC 2013; Garcia-Effron AAC 2010 C. parapsilosis not reported (intrinsic polymorphism) Prior echinocandin exposure is the key risk factor Duration of exposure (64 days median (range:3-188)) Exposure within 30 days>60 days>90 days

What is likelihood that this C. glabrata is echinocandin resistant? a. 0-5% b. 8-18% c. ~50% d. ~80% e. Almost 100% A case from UPMC C. glabrata bloodstream isolate FKS2 F659del mutant Micafungin MIC=2 µg/ml Caspofungin MIC=1 µg/ml (YeastOne)

Echinocandin susceptibility testing Reference broth microdilution (BMD) methods CLSI M27-A3 EUCAST Edef7.2 Revised Echinocandin interpretive clinical breakpoint (CBP) MICs MIC distributions Epidemiologic cut-off values Mechanisms of resistance PK/PD parameters Correlation with clinical outcomes Pfaller AJM 2011 http://www.eucast.org/fileadmin/src/media/pdfs/eucast_files/afst/eucast_edef_7_2_revision.pdf http://www.srga.org/eucastwt/bpsetting.htm

Echinocandin CBP MICs Candida spp. Agent CLSI breakpoint MICS EUCAST breakpoint MICs Susceptible Intermediate Resistant Susceptible Resistant C. albicans Anidulafungin Caspofungin Micafungin 0.25 µg/ml 0.25 µg/ml 0.25 µg/ml 0.5 µg/ml 0.5 µg/ml 0.5 µg/ml >0.5 µg/ml >0.5 µg/ml >0.5 µg/ml 0.03 µg/ml Not proposed 0.016 µg/ml >0.03 µg/ml Not proposed >0.016 µg/ml C. glabrata Anidulafungin Caspofungin Micafungin 0.125 µg/ml 0.125 µg/ml 0.06 µg/ml 0.25 µg/ml 0.25 µg/ml 0.125 µg/ml >0.25 µg/ml >0.25 µg/ml >0.125 µg/ml 0.06 µg/ml Not proposed 0.03 µg/ml >0.06 µg/ml Not proposed >0.03 µg/ml C. tropicalis, C. krusei Anidulafungin Caspofungin Micafungin 0.25 µg/ml 0.25 µg/ml 0.25 µg/ml 0.5 µg/ml 0.5 µg/ml 0.5 µg/ml >0.5 µg/ml >0.5 µg/ml >0.5 µg/ml 0.06 µg/ml Not proposed Not proposed >0.06 µg/ml Not proposed Not proposed C. parapsilosis Anidulafungin Caspofungin Micafungin 2 µg/ml 2 µg/ml 2 µg/ml 4 µg/ml 4 µg/ml 4 µg/ml >4 µg/ml >4 µg/ml >4 µg/ml 0.002 µg/ml Not proposed 0.002 µg/ml >4 µg/ml Not proposed >2 µg/ml

Reference BMD: Issue 1 Inter-laboratory variability of caspofungin MICs 24 international labs Modal variability of 4-5 dilutions in caspofungin MICs vs. C. albicans, C. glabrata, C. tropicalis and C. krusei. CLSI caspofungin CBPs could lead to reporting an excessive number of wild-type isolates as resistant Espinell-Ingroff AAC 2013

Over-calling caspofungin non-susceptibility 100 80 60 40 20 0 20 15 10 5 0 20 15 10 5 24 0 Number of Isolates Number of Isolates Number of Isolates C. glabrata (n=115) 0.06 0.12 0.25 0.5 1 2 4 C. albicans (n=39), C. krusei (n=10), C. tropicalis (n=10) 0.06 0.12 0.25 0.5 1 2 4 C. parapsilosis (n=41) Shields AAC 2012, 2013, ICAAC 2014 0.06 0.12 0.25 0.5 1 2 4

Reference BMD: Issue 2 Clinical microbiology labs do not use it Commercial antifungal susceptibility testing methods in the U.S. (CAP Proficiency testing program, 2013) 4% 12% 40% Vitek 2 12% Yeast One Other Etest Disk diffusion 32% Pfaller, CLSI 2014 Survey of 15 large U.S. tertiary care centers, 2012 53% (8/15) performed routine echinocandin susceptibility testing of Candida BSI All except one used Sensititre Yeast One Eschenauer AAC 2014

Real-world echinocandin testing 9 U.S., Australian, N.Z. hospitals, 2005-2013 Eschenauer AAC 2014

Real-world echinocandin testing 9 U.S., Australian, N.Z. hospitals, 2005-2013 Eschenauer AAC 2014 Non-susceptibility rates using CLSI CBPs Candida spp Agent(s) % Resist % Intermed. C. albicans, C. parap., C. tropicalis A, C, M 1.4% 3% C. glabrata A 6.1% 2.1% C 5.9% 17.8% M 7.5% 4.3% C. krusei A 0% 9.4% C 5.6% 46.5% M 2.5% 2.5% Most are not FKS mutants

Reference BMD: Issue 3 Unclear clinical relevance Kartsonis AAC 2005

Reference BMD: Issue 3 Unclear clinical relevance Kartsonis AAC 2005 C. albicans, krusei, tropicalis C. parapsilosis

Caspofungin CBP and C. glabrata candidemia outcomes Variable CLSI CBP >0.125 µg/ml CLSI CBP >0.25 µg/ml ROC CBP >0.5 µg/ml % sensitivity % specificity LR 95% 4% NA 95% 17% NA 60% 86% 4.3 Shields AAC 2012; ICAAC 2014 Optimal performance with Etest or YeastOne Caspofungin MIC >0.25 µg/ml Shields AAC 2013; ICAAC 2014

Risk factors for echinocandin treatment failure Variable Univariate (p) Multivariate (p) Pittsburgh Houston Pittsburgh Houston GI disease/ surgery 0.03 <0.01 0.04 Renal dysfnc N/D 0.04 Prior EC 0.008 <0.01 <0.01 Caspo MIC* 0.009 0.04 FKS mutant 0.0004 0.02 0.002 *Pitt: >0.25 µg/ml (YO) Houston: >0.125 µg/ml (YO) Shields AAC 2012 Beyda CID 2014

Algorithm for predicting C. glabrata candidemia treatment responses FKS mutants Shields AAC 2013

Algorithm for predicting C. glabrata candidemia treatment responses Shields AAC 2013

Variable Anidulafungin Micafungin MIC LR MIC LR CLSI CBP* >0.12 µg/ml 10 >0.06 µg/ml 2.4 ROC CBP >0.06 µg/ml 14 >0.03 µg/ml 4 ET, YO CBP >0.06 µg/ml 8 >0.03 µg/ml 8 *Intermediate CBP Shields AAC 2013 Our experience supports EUCAST anidulafungin and micafungin CBPs for C. glabrata

Algorithm for predicting C. glabrata candidemia treatment responses Shields AAC 2013

Conclusions (1) Echinocandins are emerging as agents of first choice against most cases of invasive candidiasis Agents are considered interchangeable Emergence of echinocandin-resistant C. glabrata with breakthrough or prior exposure (but keep it in perspective)

Conclusions (1) FKS mutations are pretty hard to induce in the clinic Never seen without prior exposure Very rarely encountered in non-c. glabrata spp. C. glabrata generally require prolonged exposure Wild-type FKS despite 80+ days of prior exposure Most treatment failures are not due to microbiologic resistance Host factors, biofilms, local PK, persistent foci, etc.

Echinocandin susceptibility testing may be a useful management tool in certain cases In patients with prior exposure, in whom you want or need to use an EC Treatment failures Routine susceptibility testing is important for epidemiologic purposes and surveillance for resistance Caspofungin MICs and CBPs by reference method are not reliable Yeast One Conclusions (1) Clinical breakpoints for all agents still need tweaking

A case from UPMC: Epilogue C. glabrata isolates from intra-abdominal abscesses (IAA) were not tested for echinocandin susceptibility at the time The second IAA isolate was shown to be FKS2 F659del mutant Micafungin MIC=2 µg/ml, Caspofungin MIC=1µg/mL (same as bloodstream isolate)

Types Types of of invasive candidiasis (IC) 1. Candidemia 40 2. Candidemia with deepseated candidiasis (DSC) Clancy and Nguyen Clin Infect Dis 2013

Types Types of of invasive candidiasis (IC) 1. Candidemia 41 2. Candidemia with deepseated candidiasis (DSC) 3. DSC without candidemia Clancy and Nguyen Clin Infect Dis 2013

What is our experience with IAC? 4.9 4.9 3.1 Types of IAC Abscess 2 peritonitis 1 peritonitis 31.9 55.2 Infected pancreas Cholangitis IAC is more common than candidemia Vergidis ICAAC 2014 Diverse types of disease Overall mortality similar to candidemia 50% of cases did not have ID consult 29% of patients do not receive timely treatment with an antifungal agent Mortality: No antifungal 37% vs. antifungal 22%

Only 3% of Candida recovered from intra-abdominal candidiasis undergo susceptibility testing Vergidis 2015 24% of patients with IAC who have received an echinocandin are infected with FKS mutant Candida Shields AAC 2014 Breakthrough: 50% Prolonged exposure (median: 119 vs 22 days) FKS mutant candidemia is often preceded by IAC MDR bacteria co-isolated in 83% of FKS mutant IAC Carbapenem-resistant K. pneumoniae Mortality rate for FKS mutant IAC 100% despite source control intervention

How can I take my clinical observations forward? Ryan Can we develop dosing regimens that optimize echinocandin efficacy and limit resistance? Paschalis Suboptimal PK at sites of IAC with conventional dosing is associated with treatment failures and resistance Can we understand the pathogenesis of IAC, and how it compares to the pathogenesis of other types of candidiasis? Candida gene expression patterns during IAC differ from other types of candidiasis, identify virulence determinants, and represent disease signatures

Cheng JID 2013 Cheng Infect Immun 2014

Site-specific caspofungin PK-PD Shields KO8 Echinocandin PK-PD within peritoneal fluid and abscesses HPLC-MS/MS PK modeling Hollow-fiber infection model in vitro to optimize PK-PD Validate regimens in mouse model Introduce optimized regimens in humans Echinocandin distribution within abscesses V. Darotis Mass spectometry imaging (MSI) New technology to directly visualize drug within infected tissues Can track unlabeled small molecules (i.e., drugs) temporally-spatially

C. albicans gene expression during IAC C. albicans nanostring nanostring profiling Probe sets included 145 C. albicans genes Control genes with high, medium and low expression Representative genes involved in antifungal responses, biofilm formation, hyphal growth, hypoxia, iron limitation, kidney infection, oxidative stress responses Transcription factors and protein kinases Peritoneal fluid harvested at 48 hours Data obtained in triplicate from 3 independently infected mice

C. albicans nanostring C. albicans gene expression during IAC Biological processes Genes Response to ph PHR1,RIM101, TEC1, ENA2, CAT1, GNP1, PHO89, RBT5, ARG1, FRP2. Response to polymorphonuclear cells DDR48, CAP1, FRP3, GCN4, TRR1, TRX1. CAT1, ENA21, GPD2, HOG1, HSP70, SSB1, Response to stress Adhesion Transporter Cellular process TRR1, TRX1, orf19.5517 ALS1, ALS3, ALS4, SAP9, TDH3, TEC1 ENA21, ENA2, GNP1, SIT1, HGT7 GCN4, GPD2

C. albicans gene expression during human IAC Drain 2--- LUQ day 1 Drain 1--- RUQ day 1, RUQ day 2 (Fluconazole)

RNA-Seq C. albicans gene expression during human IAC RNA-Seq Illumina deep-sequencing technology (Bill Nierman, JCVI) Differentially expressed genes identified using the EdgeR Bioconductor package (Camille Mesline and Nathan Clark) RNA-Seq count was normalized between the different samples and replicates using TMM A gene was considered significantly differentially expressed if its corresponding false discovery rate (FDR) was 0.001 and if its foldchange was 4

In the global comparison 3,406 genes were found to be differentially expressed between the different sample conditions at a FDR of 0.1% Replicates of each model clustered together: In vitro > ex vivo >> human and murine experiments results are reproducible for each experimental model Experiments using in vitro or ex vivo conditions are not very representative of the in vivo experiments

C. albicans C. albicans gene expression gene expression day 2 vs. day 1 RUQ-D2 vs RUQ-D1 and LUQ-D1 23% of genes were differentially expressed Up in RUQ-D2 (434 genes) Up in Day 1 samples (697 genes) Regulation of filamentous growth Adhesins, pathogenesis Ergosterol biosynthesis Nucleosome assembly, trna/ribosomal processing C3_01800C_A, CEK1, CHK1, CLA4, CLN3, CPH1, CPP1, CST20, CYR1, DEF1, EFG1, FLO8, KSP1, MDS3, MSB2, MSS11, MYO2, MYO5, NIK1, NRG1, OPY2, RFG1, RFX2, RGT1, RIM101, SET3, SFL1, SKN7, SSK1, ZCF10, ZCF16 ALS1, ALS3, CEK1, CLA4, CYR1, LPD1, NRG1, PHR2, RIM101, RVS164, etc. ERG3, ERG6, ERG10, ERG24, UPC2

In vitro Identify Candida genes that contribute to antifungal responses and pathogenesis Ex vivo Mouse or human IAC samples DC model (kidney) Identify expression signatures for diagnostics, prognosis, treatment responses or failures Profile host responses (RNA-Seq) Vergidis KL2 OPC model (tongue)

Research what you see Research what interests you clinically Once you ve asked a clinical question, you ve started a research project It s a good time to be entering I.D. research We need you Conclusions (2)

Acknowledgments UPMC Transplant ID and Antimicrobial Management Programs M. Hong Nguyen, MD (Director) Ryan Shields, PharmD Pascalis Vergidis, MD Greg Eschenauer, PharmD Brooke Decker, MD