Candida auris is an emerging multidrug-resistant yeast with high mortality. We report the sentinel C. auris case on the United States West Coast in a patient who relocated from India. We identified close phylogenetic relatedness to the South Asia clade and ERG11 Y132F and FKS1 S639Y mutations potentially explaining antifungal resistance

Crawford, Emily D

Derisi, Joseph L

Doernberg, Sarah B

Dynerman, David

Langelier, Charles

Li, Lucy M

Lyden, Amy

Miller, Steve

Nichols, Amy

Ramirez-Avila, Lynn

Serpa, Paula Hayakawa

Tato, Cristina M

Woodworth, Michael H

English

eScholarship - University of California

UCSFUC San Francisco Previously Published WorksTitleSentinel Case of Candida auris in the Western United States Following Prolonged Occult Colonization in a Returned Traveler from IndiaPermalinkhttps://escholarship.org/uc/item/98k692chJournalMicrobial Drug Resistance, 25(5)ISSN1076-6294AuthorsWoodworth, Michael HDynerman, DavidCrawford, Emily Det al.Publication Date2019-06-01DOI10.1089/mdr.2018.0408 Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaEPIDEMIOLOGYSentinel Case of Candida auris in the Western United StatesFollowing Prolonged Occult Colonization in a ReturnedTraveler from IndiaMichael H. Woodworth,1,* David Dynerman,2,* Emily D. Crawford,2 Sarah B. Doernberg,3Lynn Ramirez-Avila,4 Paula Hayakawa Serpa,2,3 Amy Nichols,5 Lucy M. Li,2 Amy Lyden,2Cristina M. Tato,2 Steve Miller,6 Joseph L. Derisi,2,7 and Charles Langelier2,3Candida auris is an emerging multidrug-resistant yeast with high mortality. We report the sentinel C. auris caseon the United States West Coast in a patient who relocated from India. We identified close phylogeneticrelatedness to the South Asia clade and ERG11 Y132F and FKS1 S639Y mutations potentially explainingantifungal resistance.Keywords: Candida auris, emerging infection, antimicrobial resistance, echinocandin resistance, metagenomicIntroductionCandida auris is an emerging fungal pathogen withhigh minimum inhibitory concentrations (MICs) for manyantifungals. Since identification in 2009, it is increasinglyrecognized as an important cause of invasive disease andnosocomial outbreaks, with high-associated in-hospitalmortality of 40–72%.1 Genomic evaluation of strainsfrom multiple geographic regions suggests simultaneousemergence of distinct geographic clades on three conti-nents, as opposed to dissemination from a single source.1This observation suggests that environmental factorssuch as increased antifungal use may have contributed toC. auris emergence.1In addition to high fluconazole MICs, C. auris isolatesalso frequently have high MICs for other antifungals, in-cluding amphotericin and less frequently echinocandins.1Despite the alarming frequency of elevated antifungal MICsin C. auris, the underlying mechanisms and alleles associ-ated with this resistance have not been fully characterized.In C. auris as well as in other Candida species, mutations inERG11 (ergosterol synthetase), FKS1 (1,3 beta-d-glucansynthetase), and FUR1 (uracil phosphoribosyltransferase)have been associated with resistance to fluconazole, echi-nocandins, and flucytosine, respectively.1,2 Previous studiessuggest that mutations in these genes can arise in the settingof systemic antifungal therapy.3Despite first appearing in the eastern United States in2013, C. auris had not been detected on the United StatesWest Coast.2 In this study, we report the identification ofC. auris in this region, which was unusual in that it did notestablish endemicity, and use whole-genome sequencing(WGS) to identify strain origin and evaluate genetic mecha-nisms of antifungal resistance.MethodsCase descriptionAn elderly man with metastatic rectal cancer relocatedfrom India to California. He had received chemother-apy and radiation while in India and had so undergoneintraabdominal surgeries complicated by sepsis. In the yearfollowing his move, he required multiple admissions to theUniversity of California, San Francisco (UCSF) Medical1Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia.2Chan Zuckerberg Biohub, San Francisco, California.3Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, San Francisco, California.4Division of Pediatric Infectious Diseases and Global Health, Department of Pediatrics, University of California, San Francisco, SanFrancisco, California.5Hospital Epidemiology and Infection Control, University of California, San Francisco, San Francisco, California.6Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California.7Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California.*These two authors contributed equally.ª Michael H. Woodworth, et al., 2019; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms ofthe Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproductionin any medium, provided the original work is properly cited.MICROBIAL DRUG RESISTANCEVolume 25, Number 5, 2019Mary Ann Liebert, Inc.DOI: 10.1089/mdr.2018.0408677Center for management of his malignancy and for secondaryinfections with carbapenem-resistant Enterobacteriaceae(CRE), for which he was placed in contact isolation. Duringhis initial multi-month admission, two cultures from his ur-ostomy grew 10,000 colony-forming units of a non-Candidaalbicans yeast that was not further speciated due to unclearclinical significance. In the course of his care, he was treatedwith echinocandins with prophylactic intent. Several monthsafter initial admission, he was transitioned to palliative care.Three days before death, a nephrostomy culture returned pos-itive for yeast, which was ultimately speciated as C. auris.Clinical microbiology and antifungalsusceptibility testingUrine collected from the patient’s nephrostomy tube intoa sterile container underwent quantitative culture for bac-teria and yeast using standard culture methods. Speciesidentification was made using matrix-assisted laser desorp-tion ionization-time of flight mass spectrometry (BruckerDiagnostics), which returned a score value of 2.14, and wasadditionally confirmed by the California Department ofPublic Health. Antifungal susceptibility testing was per-formed using Sensititre YeastOne MIC plates (Trek Diag-nostic Systems, Inc.), which has >95% agreement with theClinical Laboratory Standards Institute reference method.4Whole-genome sequencingDNA was extracted from the cultured C. auris isolate usingthe Zymo ZR Bacterial/Fungal DNA kit. Library preparationwas completed with the New England Biolabs NEBNextUltra II DNA library prep kit and WGS was performed usingan Illumina NextSeq. The same DNA also underwent libraryprep using the Oxford Nanopore Rapid Low Input by PCRBarcoding Kit and WGS on a MinION instrument.Genome assembly, phylogenetic analyses,and antifungal resistance gene analysisRaw Illumina sequencing reads were quality filtered usingPriceSeqFilter5 and then parsed with Nanopore reads for hybridde novo assembly using DBG2OLC.6 Reference-based whole-genome phylogenetic analysis constructed from core genomesingle-nucleotide polymorphisms (SNPs) was carried out withthe Northern Arizona SNP Pipeline7 using Pakistan strainB8441 as the reference genome and incorporating genomesfrom Lockhart et al.1 as well as C. auris isolate 16B15b con-taining the FKS1 S639P mutation identified by Rhodes et al.3RAxML-ng8 was used to build maximum likelihood phylo-genetic trees as detailed in Supplementary Methods (see Sup-plementary Tables S2–S4). To identify genetic mutationsassociated with fluconazole or echinocandin resistance, Illu-mina sequences were aligned against ERG11 (GenBankKY410388.1) and FKS1 (GenBank XM_018312471.1) usingBowTie2.9 Mutations were confirmed by ERG11 and FKS1PCR followed by Sanger Sequencing (SupplementaryTable S1) following previously described methods.10ResultsAssembly and phylogenetic characteristicsDe novo hybrid assembly of Illumina and Oxford Nano-pore reads produced a total of 33 contigs spanning 12 Mb,characterized by 44.9% GC content, consistent with priorestimates.1,3 Whole-genome phylogenetic analysis based ona core genome of 208,384 SNPs placed this isolate withinthe South Asia clade (Fig. 1A). On average, 56 SNPs sep-arated this isolate from others from the South Asia clade(Fig. 1B).Phenotypic and genotypic assessmentof antifungal resistanceThe California isolate demonstrated low MICs to am-photericin (1 mg/mL), flucytosine (0.5 mg/mL), and vor-iconazole (0.032 mg/mL). The isolate had an elevatedfluconazole MIC of 32 mg/mL. Assessment of this isolate’sERG11 (encoding ergosterol synthetase) allele revealed thewell-characterized Y132F substitution in the azole resis-tance hotspot region.1,3 Unlike most C. auris strains, thisCalifornia isolate also exhibited a high caspofungin MIC of8 mg/mL. Interrogation of FKS1 [encoding (1,3)-b-d-glucansynthetase] revealed a S639Y mutation in the echinocandinresistance hotspot 1 region (Supplementary Table S1).3,10DiscussionC. auris emerges on the West Coastof the United StatesIn this study, we report the first case of C. auris on theUnited States West Coast, a region that had no previousreports of the pathogen despite emergence in New York in2013. The patient’s history of health care exposure in Indiacombined with the clustering of his C. auris isolate with theSouth Asia clade by WGS phylogenetic analysis suggeststhat he acquired C. auris abroad before hospitalization inCalifornia. This finding supports current guidance from theUnited States Centers for Disease Control and Preventionto speciate all Candida in high-risk patients, includingthose from regions of high C. auris prevalence, to allow forearly implementation of infection control measures.1,11Following identification of C. auris, enhanced infectioncontrol measures were implemented at UCSF, includingsurface disinfection, a unit-level point prevalence survey,and prospective surveillance. No additional cases of C. aurisat our medical center have been identified in over a year. Thiscase represents an unusual interruption in spread and prolongedhealth care environmental contamination that has been char-acteristic of detection of health care-associated C. auris. Earlyimplementation of contact precautions for CRE may havecontributed to curbing transmission of C. auris in this case.This isolate had a high fluconazole MIC with an observedERG11 Y132F mutation.1,3 The California C. auris isolatealso demonstrated a high echinocandin MIC, which is ob-served in <10% of C. auris strains.1 It is possible that thispatient’s prophylactic treatment with echinocandins couldhave selected for resistance as observed in this isolate. ThisC. auris isolate also had a FKS1 hotspot-1 region mutation,which has been associated with echinocandin resistance inmultiple other Candida species.3,10 The identified FKS1S639 substitutions of nonpolar residues (Y, F, P) has alsobeen identified in other C. auris strains with high echino-candin MIC values, suggesting a key role for this amino acidin echinocandin resistance.3,10678 WOODWORTH ET AL.FIG. 1. (A) Phylogenetic assessment based on core genome single-nucleotide polymorphisms demonstrated the fourknown geographic clades1 and placed the California isolate within the South Asia clade. (B) Detailed phylogenetic treedescribing the South Asia clade, including the California isolate and United Kingdom outbreak isolate16B15b,3 which bothharbored the FKS1 S639P mutation. Color images are available online.679Further study is needed to estimate the prevalence andduration of colonization by this emerging pathogen. Futurework using WGS is needed to clarify the origins of C. auris,transmission patterns, and mechanisms of resistance toprevent and manage this emerging fungal pathogen of globalsignificance.Data AvailabilityRaw sequences are available via BioProject IDPRJNA480539.AcknowledgmentsThis work was supported by the National Center forAdvancing Translational Sciences (grant number TL1TR002382 to M.H.W.), the National Institute for Allergyand Infectious Disease (UM1AI104681 to M.H.W.,P01AI091575 to C.L.), the National Heart, Lung, and BloodInstitute (NHLBI K23HL138461-01A1 to C.L.), and theChan Zuckerberg Biohub ( J.L.D.). The content is solely theresponsibility of the authors and does not necessarily rep-resent the official views of the National Institutes of Health.Funding was provided by NCATS TL1 TR002382, NIAIDUM1AI104681(M.H.W.). NHLBI K12HL119997, NinaIreland Foundation, Marcus Foundation (C.L.). NIAIDP01AI091575 and the Chan Zuckerberg Biohub (E.D.C. andJ.L.D.).Disclosure StatementNo competing financial interests exist.Supplementary MaterialSupplementary DataSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4References1. Lockhart, S.R., K.A. Etienne, S. Vallabhaneni, J. Far-ooqi, A. Chowdhary, N.P. Govender, A.L. Colombo, B.Calvo, C.A. Cuomo, C.A. Desjardins, E.L. Berkow, M.Castanheira, R.E. Magobo, K. Jabeen, R.J. Asghar, J.F.Meis, B. Jackson, T. Chiller, and A.P. Litvintseva. 2017.Simultaneous emergence of multidrug-resistant Candidaauris on 3 continents confirmed by whole-genome se-quencing and epidemiological analyses. Clin. Infect. Dis.64:134–140.2. Vallabhaneni, S., A. Kallen, S. Tsay, N. Chow, R. Welsh, J.Kerins, S.K. Kemble, M. Pacilli, S.R. Black, E. Landon, J.Ridgway, T.N. Palmore, A. Zelzany, E.H. Adams, M.Quinn, S. Chaturvedi, J. Greenko, R. Fernandez, K.Southwick, E.Y. Furuya, D.P. Calfee, C. Hamula, G. Patel,P. Barrett, P. Lafaro, E.L. Berkow, H. Moulton-Meissner, J.Noble-Wang, R.P. Fagan, B.R. Jackson, S.R. Lockhart,A.P. Litvintseva, and T.M. Chiller. 2016. Investigation ofthe first seven reported cases of Candida auris, a globallyemerging invasive, multidrug-resistant fungus—UnitedStates, May 2013–August 2016. MMWR Morb. Mortal.Wkly. Rep. 65:1234–1237.3. Rhodes, J., A. Abdolrasouli, R.A. Farrer, C.A. Cuomo,D.M. Aanensen, D. Armstrong-James, M.C. Fisher, and S.Schelenz. 2018. Genomic epidemiology of the UK outbreakof the emerging human fungal pathogen Candida auris.Emerg. Microbes Infect. 7:43.4. Cuenca-Estrella, M., A. Gomez-Lopez, A. Alastruey-Izquierdo, L. Bernal-Martinez, I. Cuesta, M.J. Buitrago,and J.L. Rodriguez-Tudela. 2010. Comparison of the Vitek2 antifungal susceptibility system with the Clinical andLaboratory Standards Institute (CLSI) and EuropeanCommittee on Antimicrobial Susceptibility Testing (EU-CAST) broth microdilution reference methods and with theSensititre Ye. J. Clin. Microbiol. 48:1782–1786.5. Ruby, J.G., P. Bellare, and J.L. Derisi. 2013. PRICE:software for the targeted assembly of components of(Meta) genomic sequence data. G3 (Bethesda) 3:865–880.6. Ye, C., C.M. Hill, S. Wu, J. Ruan, and Z.S. Ma. 2016.DBG2OLC: Efficient assembly of large genomes usinglong erroneous reads of the third generation sequencingtechnologies. Sci. Rep. 6:31900.7. Roe, C., D.E. Smith, C.H.D. Williamson, M. Aziz, P. Keim,C.M. Hepp, E.M. Driebe, D. Lemmer, J. Travis, N.D.Hicks, J.M. Schupp, D.M. Wagner, D.M. Engelthaler, J.D.Gillece, J.W. Sahl, and K.P. Drees. 2016. NASP: an ac-curate, rapid method for the identification of SNPs in WGSdatasets that supports flexible input and output formats.Microb. Genomics 2:e000074.8. Kozlov, A. 2018. amkozlov/raxml-ng: RAxML-NG v0.6.0BETA (Version 0.6.0). Zenodo. http://doi.org/10.5281/zenodo.1291478.9. Langmead, B., and S.L. Salzberg. 2012. Fast gapped-readalignment with Bowtie 2. Nat. Methods 9:357–359.10. Chowdhary, A., A. Prakash, C. Sharma, M. Kordalewska,A. Kumar, S. Sarma, B. Tarai, A. Singh, G. Upadhyaya, S.Upadhyay, P. Yadav, P.K. Singh, V. Khillan, N. Sachdeva,D.S. Perlin, and J.F. Meis. 2018. A multicentre study ofantifungal susceptibility patterns among 350 Candida aurisisolates (2009–17) in India: role of the ERG11 and FKS1genes in azole and echinocandin resistance. J. Antimicrob.Chemother. 73:891–899.11. Chowdhary, A., C. Sharma, and J.F. Meis. 2017. Candidaauris: a rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally. PLoS Pathog. 13:1–10.Address correspondence to:Charles Langelier, MD, PhDDivision of Infectious DiseasesDepartment of MedicineUniversity of California, San Francisco1700 4th Street, Room 403San Francisco, CA 94158E-mail: chaz.langelier@ucsf.edu680 WOODWORTH ET AL.

Sentinel Case of Candida auris in the Western United States Following Prolonged Occult Colonization in a Returned Traveler from India

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Sentinel Case of Candida auris in the Western United States Following Prolonged Occult Colonization in a Returned Traveler from India.

Sentinel Case of Candida auris in the Western United States Following Prolonged Occult Colonization in a Returned Traveler from India.

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