Emerging Superbugs. Mark D. Gonzalez, PhD D(ABMM) Children s Healthcare of Atlanta September 7,2018. No financial disclosures

Emerging Superbugs Mark D. Gonzalez, PhD D(ABMM) Children s Healthcare of Atlanta September 7,2018 No financial disclosures

Dr Preeti Jaggi courtesy of Stan Shulman

Antibiotic Development and Resistance Antibiotic Development Antibiotic Resistance Observed Nat Chem Biol. 2007 Sep;3(9):541-8

Simoes et al. 2016. Front. Micro. 7:855.

Superbug Talk Objectives Describe their epidemiology, clinical relevance and pertinent genetic features Diagnostic issues and best practices for identification of these organisms Antimicrobial testing and treatment options/issues

Candida auris

Invasive infections are associated with high mortality (60%) Typically resistant to antifungal agents Hospital based transmission can occur, colonizes skin/mucosa and environmental surfaces Can be difficult for laboratories to identify https://www.cdc.gov/fungal/candida-auris/c-auris-drug-resistant.html

Reported Worldwide (7/31/18) First identified in 2009 https://www.cdc.gov/fungal/candida-auris/tracking-c-auris.html

361 Clinical Cases/ w/ C. auris (7/31/18) 2 1 https://www.cdc.gov/fungal/candida-auris/tracking-c-auris.html

361 Clinical Cases/ w/ C. auris (7/31/18) 2 SEACM 1 https://www.cdc.gov/fungal/candida-auris/tracking-c-auris.html

Background of C. auris

First Identification of C. auris Yeast isolated from the external ear of hospitalized patient in Japan DNA sequencing analysis 26s rdna D1/D2 domain and nuclear ribosomal DNA ITS region 2009. Microbiol Immunol 53:41-44.

26s rdna D1/D2 Phylogenetic Tree Closely related to C. haemulonii, C. pseudohaemulonii, and C. lusitaniae Known for antifungal resistance Latin for ear (auris) Spivak et al. 2017. J Clin Micro 56(2):e01588-17.

C. auris Global Emergence Slide courtesy of Dr. Carey-Ann Burnham: Jeffery-Smith et al. 2018. Clin Micro Revs. 31: e00029-17. Spivak et al. 2017. J Clin Microbiol. 56. 2: e01588-17.lockhart et al. 2017. Clin Micro Newsletter. 39-99-103. Chowdhary et al. 2017. PLOS Pathogens.

Reported Worldwide (7/31/18) Was this the spread of a single clone or distinct clones? https://www.cdc.gov/fungal/candida-auris/tracking-c-auris.html

Whole-genome sequencing analysis of 47 C. auris isolates 16 from Pakistan, 15 from India, 10 from South Africa, 5 from Venezuela and the type strain from Japan Included 2 previously sequenced genome sequences Compared the resulting sequence information 2017. Clin Infect Dis 64(2): 134-40.

Four Distinct C. auris Clades South Asia South America East Asia Clades separated by 40,000-140,000 singlenucleotide polymorphism (SNP) Within a clade, <70 SNP differences South Africa Lockhart et al. 2017. Clin Micro Newsl 39(13): 99-103.

Hunting for C. auris

Hunting for C. auris Previously misidentified yeast from 1996 in South Korea (candidemia) Examined international surveillance bank of Candida isolates (SENTRY) Contains 15,271 Candida isolates collected from 2004-2015 Identified only 4 C. auris isolates (2009, 2013, 2014 and 2015) Lee et al. 2011 J Clin Micro 49:3139-3142 Lockhart et al 2017. Clin Infect Dis 64(2): 134-40.

Reported C. auris Infections (as of Sept 2017) Type of Disease or location of Isolation No. of Reported Cases Candidemia 291 Central venous catheter tip 2 Central nervous system 1 Ear, nose and throat 21 Respiratory tract 18 Urogenital system 17 Abdominal 13 Skin and soft tissue, including surgical wounds 12 Bone 2 Adapted from Jeffrey-Smith et al 2018. Clin Micro Rev 31(1): e00029-17.

Identification Issues w/ C. auris

C. auris Identification Issues New emergence of C. auris creates a problem for identification Not included in databases Phenotypically nondescript White to cream-colored colonies on Sabouraud Pink to beige on CHROMagar Form ovoid to elongated budding yeast but typically no pseudohyphea or hyphea on corn meal or rice Tween 80 agar Grows at 37 C and 42 C

Performance of Commercial Systems Methodology API 20C BD Phoenix MicroScan Rapid Yeast Plus Vitek 2 YST* C. auris misidentified as: Rhodotorula glutinis Candida sake Candida catenulata Candida haemulonii Candida famata Candida guilliermondii Candida lusitaniae Candida parapsilosis Candida parapsilosis Candida haemulonii Candida duobushhaemulonii *biomerieux reports that updated software for Vitek 2 Yst (ver. 8.01) allows for C. auris identification Adapted from https://www.cdc.gov/fungal/candida-auris/recommendations.html

Method Used 2018 CAP F-B Mycology Proficiency Testing Survey- Yeast Identification Other BD Phoenix MicroScan Morpholgy & Biochemical Remel RapID Yeast Plus API 38.2% of labs used MALDI-TOF MS Morphology & MALDI MALDI-TOF MS Vitek 2 0 50 100 150 200 250 300 350 Number of Laboratories

Means of Identification Gene sequencing Bruker and Vitek-MS can identify Beware of the database! Updated research-use-only database for Vitek-MS (Saramis Version 4.14, Saccharomycetacea update) and Bruker (6903) and CA database

https://wwwn.cdc.gov/arisolatebank/panel/allisolate

Includes 20 yeast isolates (C. auris and related yeast) 10 C. auris isolates (diverse geographic locations) 3 C. duobushaemulonii isolates 2 C. haemulonii isolates 2 Saccharomyces cerevisiae isolates 1 Kodameae ohmeri isolate 1 C. krusei isolate 1 C. lusitaniae isolate MIC data to antifungal agents https://wwwn.cdc.gov/arisolatebank/panel/allisolate

Includes 20 yeast isolates (C. auris and related yeast) 10 C. auris isolates (diverse geographic locations) 3 C. duobushaemulonii isolates 2 C. haemulonii isolates 2 S. cerevisiae isolates 1 K. ohmeri isolate compounds 1 C. krusei may isolate not be effective) 1 C. lusitaniae isolate MIC data to antifungal agents CDC notes that these isolates should be handled with gloves in a biological safety cabinet to avoid laboratory contamination Use 10% bleach for cleaning the work area (quaternary ammonia https://wwwn.cdc.gov/arisolatebank/panel/allisolate

CDC Identification Recommendations All Candida isolates from normally sterile sites (e.g., bloodstream, CSF) should be identified to the species level ~54% of C. auris isolates are from blood Isolates from non-sterile sites should be identified: When clinically indicated for patient care When a C. auris infection/colonization is detected When a patient had an overnight stay at a healthcare facility in a country with documented C. auris infections

Lab Yeast Identification Practices Data courtesy of Dr. Carey-Ann Burnham (Washington University in St. Louis) Informal survey of 19 academic medical centers Asked about routine yeast identification practices from various body sites

Number of Laboratories Routine Yeast Identification Practices 20 15 10 5 0 Blood Sterile Body Fluids Urine Wounds Sputum BAL Genus Species Not at all Yeast Only Variable Data courtesy of Dr. Carey-Ann Burnham

Antifungal Susceptibility

Antifungal Susceptibility Testing Currently no species specific breakpoints for C. auris CDC recommends performing antifungal susceptibility testing on all C. auris isolates CDC has tentative breakpoint information Correlation between breakpoints and clinical outcomes is unknown

CDC Tentative Breakpoints Drug Fluconazole Voriconazole Posaconazole Amphotericin B Tentative MIC Breakpoint (µg/ml) 32 N/A 2 Comment Modal MIC was 256; isolates w/ MIC of 32 had a resistance mutation in Erg11 gene Consider fluconazole susceptibility as surrogate for 2 nd generation triazoles; Isolate that fluconazole resistant may still respond to other triazoles Based on pharmoacokinetic/pharmacodynamic analysis in mouse model w/ standard dosing Caspofungin 2 Tentative breakpoints based on modal MIC Anidulafungin distribution of ~100 isolates from diverse 4 Micafungin geographic locations Adapted from https://www.cdc.gov/fungal/candida-auris/recommendations.html

C. auris Antifungal MIC Data Spivak et al. 2017. J Clin Micro 56(2):e01588-17.

Additional Susceptibility Notes Tentative breakpoints are not established Not definitive Data could change Reports of falsely elevated amphotericin and caspofungin MICs with the Vitek 2 yeast susceptibility testing method

Antifungal Resistance Determinants Studies in C. albicans indicate that resistance occurs through overexpression of transporters, mutations in target gene and target protein overexpression Target site mutations known to confer fluconazole resistance in C. albicans have been identified in C. auris isolates C. auris genome contains numerous predicated transporters

Treatment- CDC Recommendations Age Group Adults and children 2 months of age Neonates and infants < 2 months of age Initial Antifungal Treatment Echinocandin; Patients should be carefully monitored for clinical improvement, and follow-up cultures (repeat susceptibility testing) Amphotericin B deoxycholate, if unresponsive then liposomal amphotericin B Consultation with infectious disease specialist is highly recommended CDC does not recommend treating C. auris isolates from noninvasive sites when there is no evidence of infection https://www.cdc.gov/fungal/candida-auris/c-auris-treatment.html

Infection Prevention

Infection Prevention Transmission within hospitals of C. auris has been documented C. auris can colonize the skin and mucosa and environmental surfaces Contact mediated transmission seems quite probable

CDC Recommendations Patients with C. auris should be in a singlepatient room with standard and contact precautions Continue precautions for as long the patient is colonized (months?) Emphasis on hand hygiene Alcohol-based hand sanitizers can be used If hands visibly soile, wash w/ soap and water

CDC Recommendations Cleaning and disinfection of patient care environment Use EPA-registered hospital-grad disinfectant effective against Clostridioides difficile spores Thorough daily and terminal cleaning Screening contacts of identified case patients for C. auris colonization

CDC Screening Recommendations Axilla and groin appear to be the most commonly colonized site (although also found in nose, external ear canal, oropharynx, urine, wounds and rectum) Composite swab of the axilla and groin Contact local public health lab to coordinate with CDC and AR Lab Network

C. auris Final Comments As more information is being gained, changes will occur CDC website is a great resource (link below) Contact CDC at candidaauris@cdc.gov https://www.cdc.gov/fungal/candida-auris/index.html

Carbapenem-resistant Enterobacteriaceae (CRE)

CRE are an categorized as an urgent threat by the CDC Along w/ β-lactam resistance, resistance to other drug classes can occur https://www.cdc.gov/drugresistance/biggest_threats.html

Current CDC Definition for CRE (released in 2015) Resistance to doripenem, ertapenem, imipenem, or meropenem OR documentation of carbapenemase production Requires use of the current CLSI carbapenem breakpoints Can be a general description that does not describe the mechanism

Current CDC Definition for CRE (released in 2015) Resistance to doripenem, ertapenem, imipenem, or meropenem OR documentation of carbapenemase production Proteus Requires spp., use Providencia of the current spp. CLSI and carbapenem Morganella spp. breakpoints may have elevated minimal MICs to imipenem (not to be considered CRE) Can be a general description that does not describe the mechanism

CRE Non-carbapenemase - Non-CP-CRE - Cephalosporinase (ESBL, AmpC) - Porin, pump upregulation Carbapenemase - CP-CRE - β-lactamase that degrades carbapenems - IMP, KPC, NDM, OXA, VIM

Gram-Negative Cell Envelope Non-Carbapenemase Porins Cephalosporinase Pumps Lister et al 2009. CMR. 22(4):582-610.

Gram-Negative Cell Envelope Carbapenemase Carbapenemase Lister et al 2009. CMR. 22(4):582-610.

Carbapenemase Classification Class Carbapenemase Enterobacteriaceae Nonfermenters A (Serine) KPC 1 +++ + B (metallo-βlactamase) NDM 2, IMP, VIM +++ +++ D (Serine) OXA +++ +/- 1 Most common in USA 2 Increasing prevalence in USA Adapted from slide courtesy of Janet Hindler

Patients w/ KPC-producing CRE as of December 2017 Patients w/ NDM-producing CRE as of December 2017 https://www.cdc.gov/hai/organisms/cre/trackingcre.html

https://www.cdc.gov/hai/organisms/cre/trackingcre.html Patients w/ IMP-producing CRE as of December 2017 Patients w/ OXA-48 type producing CRE as of December 2017

Patients w/ VIM-producing CRE as of December 2017 https://www.cdc.gov/hai/organisms/cre/trackingcre.html

Old vs Revised Carbapenem Breakpoints Agent Old CLSI Breakpoints (pre-2010) Revised CLSI Breakpoints (2010) S I R S I R Doripenem None 1 2 4 Ertapenem 2 4 8 0.5 1 2 Imipenem 4 8 16 1 2 4 Meropenem 4 8 16 1 2 4 Perform carbapenemase test if ertapenem MIC=2 and meropenem/ imipenem MIC=2-4 If carbapenemase positive, change susceptible to resistant Perform carbapenemase test for epi or infection prevention, if requested Report results/interp. as tested (even if carbapenemase) FDA Enterobacteriaceae carbapenem breakpoints now match CLSI

https://www.fda.gov/drugs/developmentapprovalprocess/developmentresources/ucm575163.htm

Vast majority of laboratories use commercial automated antimicrobial susceptibility test (AST) systems Revised carbapenem breakpoints requires laboratories to perform verification studies 2018. Clin Infect Dis 66(7):1061-7.

Survey of California Laboratories Conducted Fall of 2015 to Spring 2016 264 (67%) of 392 hospitals and long-term acute care hospitals responded 129 unique clinical microbiology laboratories (1 lab did not perform AST, 2 did not disclose AST method) Data from 128 laboratories Humphries et al 2018. Clin Infect Dis 66(7):1061-7.

California Survey Results Test System Number of Labs # Using Revised Breakpoints (%) Vitek 72 48 (67) MicroScan 37 24 (65) Phoenix 10 10 (100) Sensititre 2 2 (100) Manual 5 5 (100) 92 (72%) of 128 laboratories use the revised CLSI breakpoints Humphries et al 2018. Clin Infect Dis 66(7):1061-7.

California Survey Results 38 labs were not compliant w/ CLSI guidance 35 labs using current breakpoints but modifying carbapenem results if carbapenemase positive 3 labs using old breakpoints but did not modify carbapenem results if carbapenemase positive Humphries et al 2018. Clin Infect Dis 66(7):1061-7.

California Time to Implementation 74 laboratories disclosed the time to implementation Average 41 months, median 55 months Humphries et al 2018. Clin Infect Dis 66(7):1061-7.

Georgia CRE Survey Data-Fall 2017 Data courtesy of Jeanne Negley, Elizabeth Smith and Chris Bower Response from 80 Georgia laboratories

Georgia Lab Results Not indicated Referred, not performed, not known Old breakpoints 13.8% Revised breakpoints 65% Number of Responding Laboratories

Simulation model 32 month delay in adopting revised carbapenem breakpoints is associated w/ 1821 additional CRE carriers (carbapenemase) in Orange County, California 2016. J Clin Micro 54(11):2757-62.

What resources are available to validate the revised carbapenem breakpoints?

Collection of 31 Enterobacteriaceae isolates Variable carbapenem MICs and carbapenem resistance mechanisms Includes Citrobacter spp (n=5), Enterobacter spp. (n=5), Escherichia coli (n=9), Klebsiella spp (n=7), Proteus mirabilis (n=1), Providencia stuartii (n=1), Salmonella spp (n=1), Serratia marcescens (n=1) and Shigella sonnei (n=1) https://wwwn.cdc.gov/arisolatebank/panel/allisolate

Isolate Example https://wwwn.cdc.gov/arisolatebank/panel/allisolate

Revising Breakpoints Isolates of defined susceptibility and diverse profiles (CDC bank) CLSI M52 document and CLSI website Can work with the manufacturer to revise breakpoint interpretations Education to physicians about the change

Methods for Identifying Carbapenemase Producers

Carbapenemase Detection Phenotypic Demonstration of carbapenemase activity in vitro Genotypic methods Molecular detection of carbapenemase genes

CLSI Carbapenemase Detection Methods Organisms CarbaNP Enterobacteriaceae P. aeruginosa Strengths Rapid Limitations -Special reagents -Invalid results occur -Certain carbapenemases are missed (OXA-type, chromosomally encoded) Adapted from Table 3B and 3C CLSI M100-S28

CLSI Carbapenemase Detection Methods Organisms CarbaNP Enterobacteriaceae P. aeruginosa mcim Enterobacteriaceae P. aeruginosa Strengths Rapid No special reagents or media needed Limitations -Special reagents -Invalid results occur -Certain carbapenemases are missed (OXA-type, chromosomally encoded) Overnight incubation Adapted from Table 3B and 3C CLSI M100-S28

CLSI Carbapenemase Detection Methods Organisms CarbaNP mcim mcim w/ ecim Enterobacteriaceae P. aeruginosa Enterobacteriaceae P. aeruginosa Enterobacteriaceae positive by mcim Strengths Rapid No special reagents or media needed Limitations -Special reagents -Invalid results occur -Certain carbapenemases are missed (OXA-type, chromosomally encoded) Overnight incubation No special reagents or media needed Overnight incubation Adapted from Table 3B and 3C CLSI M100-S28

CLSI Carbapenemase Detection Methods Organisms Strengths Limitations Other (e.g., Molecular) Enterobacteriaceae P. aeruginosa Determine mechanism for CarbaNP or mcim positives Determines type of carbapenemase (if present) -Special reagents and equipment -Specific to targeted gene, false-negatives can occur Adapted from Table 3B and 3C CLSI M100-S28

Modified Hodge Test for Carbapenemase Detection + - + - + - No longer recommended by CLSI In California study, 84% of laboratories that perform carbapenemase testing in-house, use modified Hodge test

Carba NP Assay Suspension of Enterobacteriaceae or P. aeruginosa isolate of interest in a protein extraction solution Incubated in presence of imipenem with phenol red as a ph indicator Hydrolysis of imipenem results in a drop in ph (color change)

Carba NP Assay Results

Modified Carbapenem Inhibition Method (mcim) Phenotypic test for Enterobacteriaceae or P. aeruginosa isolate of interest Inactivation of meropenem present within a disk

mcim Procedure Incubate for 4 hours at 35 C Zone diameter of 6-15 mm Incubate for 18-24 hours at 35 C Zone diameter of 19 mm van der Zwaluw et al. 2015. PLoS ONE 10(3): e0123690.

ecim Phenotypic test for Enterobacteriaceae that are positive in the mcim assay Determine if carbapenemase is a metallo-βlactamase Chelating metal with EDTA

ecim results Metallo-β-lactamase Detected mcim ecim Increased zone by 5 mm mcim Serine carbapenemase Detected ecim Increased zone by 4 mm

Collection of 80 isolates Variable carbapenem MICs and carbapenem resistance mechanisms Includes Acinetobacter baumannii (n=14), Enterobacteriaceae (n=54), and Pseudomonas aeruginosa (n=12) https://wwwn.cdc.gov/arisolatebank/panel/allisolate

FDA-Cleared Genotypic Testing Methods Method Cepheid CarbaR FilmArray BCID Panel Verigene Gram- Negative Blood Culture Test Specimen Type Cultured isolates, rectal/peri rectal swabs Positive blood culture broth Positive blood culture broth Gene(s) Detected bla IMP bla KPC bla NDM bla OXA-48 bla VIM bla KPC bla IMP bla KPC bla NDM bla OXA bla VIM Gene detection occurs in isolates of Acinetobacter baumannii, Enterobacteriaceae and Pseudomonas aeruginosa

Potential β-lactam Antibiotics for Carbapenemase Producers Antibiotic Organisms Enzymes Breakpoints Ceftazidime-avibactam Meropenem-vaborbactam Enterobacteriaceae P. aeruginosa Enterobacteriaceae KPC, not metallos KPC, not metallos CLSI FDA FDA

Infection Prevention

Newly identified CRE-colonized or CREinfected patient Notify appropriate staff Notify public health (if required) Place patient on Contact Precautions in single room Reinforce hand hygiene and use of Contact Precautions Consider screening epidemiologically-linked patient contacts for CRE Antimicrobial resistance (AR) network and your local public health lab can help with screening cultures Adapted from https://www.cdc.gov/hai/pdfs/cre/cre-guidance-508.pdf

Summary Diversity of pathogens and antimicrobial resistance are changing Limitations of current testing methodologies need to be acknowledged and changed if possible

Questions?