Simultaneous Emergence of Multidrug-Resistant Candida auris on 3 Continents Confirmed by Whole-Genome Sequencing and Epidemiological Analyses
Simultaneous Emergence of Multidrug-Resistant Candida auris on 3 Continents Confirmed by Whole-Genome Sequencing and Epidemiological Analyses
Shawn R. Lockhart
Kizee A. Etienne
Nelesh P. Govender
Arnaldo Lopes Colombo
Christina A. Cuomo
Christopher A. Desjardins
Elizabeth L. Berkow
Rindidzani E. Magobo
Rana J. Asghar
Jacques F. Meis
Anastasia P. Litvintseva
Background. Candida auris, a multidrug-resistant yeast that causes invasive infections, was first described in 2009 in Japan and has since been reported from several countries. Methods. To understand the global emergence and epidemiology of C. auris, we obtained isolates from 54 patients with C. auris infection from Pakistan, India, South Africa, and Venezuela during 2012-2015 and the type specimen from Japan. Patient information was available for 41 of the isolates. We conducted antifungal susceptibility testing and whole-genome sequencing (WGS). Results. Available clinical information revealed that 41% of patients had diabetes mellitus, 51% had undergone recent surgery, 73% had a central venous catheter, and 41% were receiving systemic antifungal therapy when C. auris was isolated. The median time from admission to infection was 19 days (interquartile range, 9-36 days), 61% of patients had bloodstream infection, and 59% died. Using stringent break points, 93% of isolates were resistant to fluconazole, 35% to amphotericin B, and 7% to echinocandins; 41% were resistant to 2 antifungal classes and 4% were resistant to 3 classes. WGS demonstrated that isolates were grouped into unique clades by geographic region. Clades were separated by thousands of single-nucleotide polymorphisms, but within each clade isolates were clonal. Different mutations in ERG11 were associated with azole resistance in each geographic clade. Conclusions. C. auris is an emerging healthcare-associated pathogen associated with high mortality. Treatment options are limited, due to antifungal resistance. WGS analysis suggests nearly simultaneous, and recent, independent emergence of different clonal populations on 3 continents. Risk factors and transmission mechanisms need to be elucidated to guide control measures.
C. auris exhibits resistance to fluconazole and variable
susceptibility to other azoles, amphotericin B, and echinocandins.
It phenotypically resembles Candida haemulonii and requires
use of molecular methods for identification [9, 10]. C. auris
can be a challenge to identify and treat, especially in
resourcelimited settings, where molecular identification may not be
immediately available and access to antifungals other than
fluconazole may be limited.
In 2015, the US Centers for Disease Control and Prevention
(CDC) was asked to assist with an outbreak of BSIs and positive
urine cultures caused by presumed Saccharomyces at a
hospital in Pakistan. The isolates were shipped to the CDC, where
they were identified as C. auris, which had not previously been
reported from Pakistan. At the same time, the CDC was aware
of increasing numbers of cases of C. auris infections in both
India and South Africa [4, 6, 7] and was subsequently notified
of cases from Venezuela . An international collaboration
was established to better understand the epidemiology of C.
auris, determine the extent of resistance and whether the
emergence of this organism was occurring independently in
multiple countries or was caused by the spread of a single outbreak
strain. Here we report clinical characteristics of patients with
C. auris infection, antifungal susceptibility patterns, and results
of whole-genome sequence (WGS) analysis of C. auris isolates.
Case Patient Information
Case patient information was obtained using a standardized
surveillance case report form and was available for 41 (76%) of the 54
patients with isolates. The CDC’s National Center for Emerging
and Zoonotic Infectious Diseases determined that this project
constituted a nonresearch public health surveillance activity.
Fifty-four isolates from 54 patients during 2012–2015 were
collected from Pakistan (n = 18; 2 hospitals), India (n = 19; 3
hospitals), South Africa, (n = 10; 8 hospitals), and Venezuela (n = 5;
1 hospital) (Supplemental Table 1). In addition, the type
specimen from Japan was included as was a retrospectively identified
Pakistan isolate from 2008. Isolates were from blood (n = 27),
urine (n = 10), soft tissue (n = 5), or other sites (n = 12).
DNA Purification and Isolate Identification
DNA was extracted using the ZYMO Research ZR Fungal/
Bacterial DNA MiniPrep kit . Species identities were
confirmed by sequencing the D1–D2 region of the 28S subunit of
ribosomal DNA .
Antifungal Susceptibility Testing
Antifungal susceptibility testing was performed as described
elsewhere . C. auris–specific break points were defined
conservatively based on those established for closely related
Candida species. Because the modal minimum inhibitory
concentration (MIC) to fluconazole was at the upper limit of the
measured distribution, resistance to fluconazole was
arbitrarily set at ≥32 µg/mL. Other break points were ≥2 µg/mL for
voriconazole (as for Candida krusei ), ≥8 µg/mL for the
echinocandins (as for Candida parapsilosis and Candida
guilliermondii ), ≥128 µg/mL for flucytosine (above the
achievable dose), and ≥2 µg/mL for amphotericin B.
WGS, Genomic Assembly, and Single-Nucleotide Polymorphism Identification
Of 54 available isolates, 47 were selected for WGS. To improve
assembly, 2 isolates were sequenced using the PacBio
platform, and all 47 isolates were sequenced using Illumina HiSeq.
Sequencing libraries were prepared as described elsewhere .
Two previously published genomes of C. auris (ERR899743 and
SRR1664627) from India were included [16, 17]. The reference
genome was generated by de novo assembly of PacBio reads
from isolate B8441, as described elsewhere , and used as
a reference for single-nucleotide polymorphism (SNP)
calling. SNPs were identified using 2 independent pipelines, as
described elsewhere (see also the legend to Supplemental Figure
1) [15, 19]. The SNP calls were filtered and included in the final
matrix if they were not identified in repetitive regions, were
found in <90% of the base calls at that position, and had a
minimum read depth coverage of 10. Reads that mapped to multiple
locations and indels were excluded. Genome-wide SNP-based
phylogenetic analyses was conducted using the maximum
parsimony algorithm in MEGA V6.06 software, . Statistical
significance of the phylogenetic tree was tested using bootstrap
analysis with 1000 reiterations. Read data were deposited into
the National Center for Biotechnology Information’s Sequence
Read Archive under BioProject PRJNA328792.
Azole Resistance Mutation Identification
For resistance gene analysis, orthologous sequences to Candida
albicans ERG11 from SC5314 Assembly 22 (Candida Genome
Database; http://www.candidagenome.org/) were extracted
from each C. auris genome and aligned using a ClustalW
alignment in MEGA V6.06 software . Sequences were
evaluated for amino acid substitutions that corresponded to those
described elsewhere within hot-spot regions in azole-resistant
C. albicans .
Query of a Global Candidemia Surveillance Program for C. auris
To understand whether C. auris emerged after 2009 or had been
overlooked or misidentified in the past, we queried an
ongoing international antifungal surveillance program, SENTRY
(JMI Laboratories)  containing 15 271 candidemia
isolates collected from 152 international medical centers during
2004–2015, including from Asia (n = 41), Europe (n = 50),
Latin America (n = 15), and North America (n = 46). Isolate
identification from 2004 to 2009 was confirmed using a
combination of morphological and biochemical tests. After 2009,
all uncommon Candida species were subject to DNA
sequencing and/or matrix-assisted laser desorption/ionization-time of
flight (MALDI-TOF) mass spectrometry . Four presumed
C. haemulonii (the most common misidentification of C. auris)
isolates collected before 2009 were retrospectively reidentified
as C. auris by using MALDI-TOF mass spectrometry.
Case Patient Description
Demographic and clinical information was available for 41
(76%) of 54 patients (Table 1). Nearly half (44%) of the patients
with clinical information were from Pakistan (n = 18), 15 (37%)
were from India, 5 (12%) were from Venezuela, and 3 (7%) were
from South Africa. The median age was 54 years
(interquartile range [IQR], 24–69); 3 patients, all from Venezuela, were
Patients, No. (%)a
Abbreviation: IQR, interquartile range.
aData represent No. ( %) of patients unless otherwise specified.
neonates (≤28 days old); 26 (63%) were male. Diabetes mellitus
was the most common underlying condition identified. Half of
all patients (n = 21; 51%) had undergone surgery in the 90 days
before having a culture yielding C. auris; the most common
operations included abdominal surgery (n = 6) diabetic limb
amputations (n = 5), and cardiac surgery (n = 3). Seventy-three
percent (n = 30) had a central venous catheter, and 61% (n =
25) had a urinary catheter. Forty-one percent of patients had
received systemic antifungal therapy (primarily fluconazole) in
the 90 days before C. auris infection. The median time from
admission to diagnosis was 19 days (IQR, 9–36 days).
Fifteen patients did not receive any antifungal treatment
and 7 (47%) of those died including 4 of the 5 patients with
BSIs who received no treatment. Twenty-six patients received
antifungal treatment, and 6 received ≥2 antifungals. Three
patients received fluconazole alone, 1 of whom had a BSI and
died. Fifteen patients received amphotericin B, 4 of whom had
isolates resistant to amphotericin B; 2 patients with
amphotericin B–resistant isolates survived while the remaining 2 died.
Eight patients were treated with caspofungin, including 1 whose
isolate was resistant to caspofungin; 4 of these patients died,
including the patient with the echinocandin-resistant isolate.
Overall, 59% (n = 24) of the patients died. Of the 25
patients with BSIs, 17 (68%) died. Of the 7 patients with
urinary tract infections, 5 (71%) died, most likely of associated
sepsis. Because of the seriousness of the underlying disease of
many of the patients, whether and how much C. auris
attributed to death could not be determined.
Table 2. Antifungal Susceptibility Data for 54 Candida auris Isolates
MIC Range, µg/mL
Abbreviations: MIC, minimum inhibitory concentration; MIC50, MIC for 50% of isolates;
MIC90, MIC for 90% of isolates.
Antifungal Susceptibility Testing
Antifungal susceptibility testing was performed on 54 isolates.
The MIC range and the MICs for 50% and 90% of isolates
are shown in Table 2, and the MIC distribution is shown in
Supplemental Table 2. Using stringent break points, 50 isolates
(93%) were resistant to fluconazole, 29 (54%) to voriconazole
(≥2 µg/mL), 19 (35%) to amphotericin B (7 from Pakistan and
12 from India), 4 (7%) to echinocandins (2 from India and 2
from South Africa), and 3 (6%) (from India) were resistant
to flucytosine. Two isolates, both from India, were resistant
to fluconazole, voriconazole, echinocandins, and
amphotericin B. In all, 22 (41%) isolates were resistant to ≥2 classes
ERG11 Mutation Analysis
By comparing Erg11 amino acid sequences between C.
albicans and C. auris, 9 amino acid substitutions, which have been
identified in resistant but not wild-type C. albicans isolates,
were identified in all C. auris isolates. Three additional hot-spot
amino acid substitutions were identified that have been either
proposed or proved to significantly increase fluconazole
resistance in C. albicans [23, 24]. These substitutions were strongly
associated with geographic clades: F126T with South Africa,
Y132F with Venezuela, and Y132F or K143R with India and
Pakistan (Figure 1, Supplemental Table 3).
WGS was performed on 47 isolates, including 16 from Pakistan,
15 from India, 10 from South Africa, 5 from Venezuela, and
the type specimen from Japan; 2 previously sequenced genomes
from National Center for Biotechnology Information’s Sequence
Read Archive were also included. The assembled genome size
was 12.5 Mb, similar to that of other Candida species .
Reference-based phylogenetic analysis using the NASP
pipeline identified approximately 119 000 shared SNPs, of which
86% were parsimoniously informative. Phylogenetic analysis
identified a strong phylogeographic structure comprising 4
distinct clades, which were separated by tens of thousands of SNPs
and represented distinct geographic regions: South Asia (India/
Pakistan), South Africa, South America (Venezuela), and East
Asia (Japan) (Figure 1). Far fewer SNPs were identified within
each cluster; for example, <16 SNPs differentiated any 2 isolates
from the South American clade, <70 SNPs differentiated any 2
isolates from South Africa, and <60 SNPs differentiated 34 of 36
isolates from South Asia. Two isolates, B8441, the Pakistan
isolate from 2008, and B11112, also from Pakistan, differed from
the rest of the South Asian strains by >600 SNPs. Furthermore,
within the South Asian clade, 2 smaller clusters were
identified. These clusters were associated with a single hospital in
Pakistan and consisted of nearly identical strains (differences of
<2 SNPs). Comparable results were obtained using the GATK
pipeline for SNP calling (Supplemental Figure 1).
In an effort to test whether C. auris had been misidentified or
overlooked before the recent emergence, we queried the
international surveillance program SENTRY, which contained 15 271
Candida isolates collected from 2004 to 2015. Four isolates were
identified as C. auris (from 2009, 2013, 2014, and 2015), 1 of
which had been previously identified as C. haemulonii, further
supporting the scarcity of C. auris before 2009.
C. auris is a globally emerging multidrug-resistant yeast that
can cause invasive infections. Here we report C. auris infections
from Pakistan, adding to previous reports from Japan, South
Korea, India, South Africa, Venezuela, and Kuwait. Based on
the large number of SNPs observed across 47 isolates from 4
regions (South Asia, East Asia, South America, and South
Africa) and minimal intraregion genetic diversity, WGS
analysis suggests near-simultaneous emergence of C. auris in ≥4
locations rather than recent spread from a single source [1, 2, 4,
7, 8]. Although the causes for such emergence are not clear, they
may include new or increasing antifungal selection pressures in
humans, animals, or the environment.
One possible explanation for the apparent recent emergence of
C. auris may be that this pathogen has not been previously
recognized. To test this hypothesis, we performed a literature review
and queried the available global culture collections. Although the
first publication of C. auris was from 2009, the earliest reported
C. auris isolate was found in a Korean isolate collection,
having come from a 1996 BSI in a pediatric surgery patient .
Furthermore, we identified a 2008 C. auris isolate from Pakistan,
which had not been previously recognized . However, to our
knowledge, no other C. auris isolates from 1996–2009 have been
reported. Our retrospective review of the SENTRY isolate
collection, with 15 271 isolates of Candida from 4 continents, did not
find C. auris isolates before 2009 confirming that this pathogen
was not simply misidentified previously and was indeed rare.
Examination of additional isolate collections from around the
world would help to further validate this conclusion.
Increased clinical availability of antifungal agents may have
contributed to the emergence of this organism. Amphotericin B
has been available since 1954, fluconazole since 1991, and
echinocandins since the early 2000s, but access to these drugs occurred
much later in resource-limited settings. Although accurate data
on the antifungal prescription practices are difficult to obtain,
anecdotal evidence suggests increased use in recent years of
triazoles and other antifungals for empiric treatment of surgical
and other hospitalized patients. In South Africa, C. auris seems
to have emerged more rapidly in private-sector hospitals where
echinocandin use is much higher than in public-sector
hospitals . Two of the echinocandin-resistant isolate came from
South Africa, although neither patient received an
echinocandin, suggesting exogenous spread between patients. The fact that
a substantial proportion of patients in this investigation were
receiving antifungal treatment when C. auris infection was
diagnosed supports the hypothesis that antifungal selection pressure
may, in part, be responsible for the emergence of C. auris.
C. auris is phylogenetically related to Candida species
krusei, lusitaniae, and haemulonii, which are known to have either
intrinsic or inducible resistance to fluconazole ,
amphotericin B , or both . The C. auris isolates in the current
study exhibit very high MICs for fluconazole, and all but 4
isolates carried amino acid substitutions shown to significantly
increase fluconazole resistance . There were no isolates with
high MICs (>2 µg/mL) for either itraconazole or posaconazole.
Several isolates were shown to have in vitro resistance to the
2 remaining major classes of antifungals, polyenes and
echinocandins, suggesting that resistance may be inducible under
antifungal pressure. This finding poses an important clinical
challenge. A small proportion of isolates demonstrated elevated
MICs for all 3 major antifungal classes, severely limiting
WGS analysis demonstrated low genetic diversity among
isolates within each clade; even across the largest clade of 36 isolates
from India and Pakistan, all but 2 isolates differed by <60 SNPs,
despite the fact that isolates came from 5 hospitals, thousands
of miles apart. A recent WGS study from India with 5 isolates
demonstrated similar results . Clonality within C. auris has
been shown elsewhere using amplified fragment length
polymorphism, multilocus sequence typing, and MALDI-TOF mass
spectrometry using some of the same Indian isolates from our
study [4, 6]; however, the low discriminatory power of those
methods did not allow an assessment of genetic relatedness
between isolates with identical genotypes [4, 6, 29, 30]. Our
WGS analysis shows that the isolates from different regions
differed by tens of thousands of SNPs; however, the mean number
of SNPs within each geographic cluster was minimal (<70 for
isolates within each cluster), again strongly suggesting recent
Our results raise concern that C. auris may spread within the
hospital setting, evidenced by the presence of nearly identical
isolates in 2 hospitals in South Asia. The fact that a majority
of patients had a central venous catheter, a urinary catheter,
or a recent surgical procedure as possible site of entry, and the
timing of infection, a median of 19 days after admission, also
support this hypothesis. Like other Candida infections, C. auris
infections seem to be hospital acquired and occur several weeks
into a patient’s hospital stay, suggesting an exogenous rather
than endogenous source and a breach of infection control
measures. The overall in-hospital mortality rate of 60% is
similar to or higher than those reported from these countries or
regions: 52% in Pakistan , 44% in India , 46% in South
Africa , and 72% in South America . We were unable to
examine time to clearance of infection; however, another study
found persistent fungemia up to 3 weeks after initiation of
antifungal treatment .
The current study has a number of limitations. First, only
limited clinical data were available for each patient, and
clinical data were not available for all patients whose isolates
were included in the study. Second, we were unable to
identify risk factors that were specific to C. auris because we did
not have a comparison group of patients with other Candida
infections from the same institutions . Third, because
there are no universally accepted break points for C. auris,
antifungal resistance was inferred based on break points
for other species. Fourth, extremely low genetic diversity
of C. auris strains hampered our ability to understand the
movement of isolates within each geographic area. Although
WGS of isolates from a Pakistani hospital identified 2
subclusters of nearly identical isolates, which was consistent
with an ongoing nosocomial outbreak, the exceptionally low
diversity among other South Asian isolates made it difficult
to establish a relevant cutoff number of SNPs that would
differentiate this institutional outbreak from other isolates
circulating in the region.
C. auris has been documented in numerous countries on 3
continents in the past 7 years. It is likely that there are other
places where this organism is already circulating but has not
yet been identified or reported, and it will probably emerge in
new locations as well. Multidrug resistance and high
associated mortality rates makes C. auris an emerging global threat.
Of additional concern, C. auris may become widely established
within certain regions, as evidenced by the fact that it now
accounts for nearly 5% of candidemia in Indian intensive care
units . There are still many unanswered questions about
C. auris, including why it has suddenly emerged, whether its
clonal expansion and global distribution will level off or
continue, how it is transmitted, and what infection prevention and
control measures are needed to prevent its spread within
hospitals. Further research on risk factors for this infection, how
it is acquired and transmitted, and how the fungus develops
resistance are needed to control the spread of this pathogen.
Supplementary materials are available at Clinical Infectious Diseases online.
Consisting of data provided by the author to benefit the reader, the posted
materials are not copyedited and are the sole responsibility of the author, so
questions or comments should be addressed to the author.
Acknowledgments. We thank Lalitha Gade, Joyce Peterson, Carol
Bolden, Randal Kuykendall, and Colleen Lysen at the Centers for Disease
Control and Prevention for assistance with processing and identification of
the isolates; Mike Frace from the Centers for Disease Control and Prevention
(CDC) DNA Core Facility for WGS sequencing and PacBio assembly; Afia
Zafar, Salima Qamar, Fizza Farooqui, Yusra Riyasat, Faisal Mahmood,
Nosheen Nasir, and Rozina Roshan from Aga Khan University Hospital;
Shamoona Fareeha Ather and Asad Soomro from the Field Epidemiology
and Laboratory Training Program in Pakistan; Craig Corcoran from
Ampath Laboratories for sharing South African isolates; and Erika Britz
and Verushka Chetty at the National Institute for Communicable Diseases
for assistance with data collection.
Disclaimer. The contents of this publication are solely the
responsibility of the authors and do not necessarily represent the official views of
the National Institutes of Health or the Centers for Disease Control and
Financial support. This work was supported by the National Institute
of Allergy and Infectious Diseases (grant U19AI110818 to the Broad
Institute), United States Agency for International Development (USAID;
grant 4-338/PAK-US/HEC/2010/932 to Aga Khan University), and the
Advanced Molecular Detection initiative at the CDC.
Potential conflicts of interest. N. P. G. has received speaker honoraria
from Pfizer, Astellas, and MSD, travel grants from MSD, has provided
educational materials for TerraNova, and has acted a temporary consultant for
Fujifilm Pharmaceuticals. A. L. C. has received educational grants from
Pfizer, Gilead, United Medical, and Astellas and funding for research from
Pfizer and Astellas. M. C. is an employee of JMI Laboratories, which has
received research and educational grants in 2014–2015 from Achaogen,
Actavis, Actelion, Allergan, American Proficiency Institute, AmpliPhi,
Anacor, Astellas, AstraZeneca, Basilea, Bayer, BD, Cardeas, Cellceutix,
CEM-102 Pharmaceuticals, Cempra, Cerexa, Cidara, Cormedix, Cubist,
Debiopharm, Dipexium, Dong Wha, Durata, Enteris, Exela, Forest Research
Institute, Furiex, Genentech, GlaxoSmithKline, Helperby, Institute for
Clinical Pharmacodynamics, Janssen, Lannett, Longitude, Medpace, Meiji
Seika Kasha, Melinta, Merck, Motif, Nabriva, Novartis, Paratek, Pfizer,
Pocared, PTC Therapeutics, Rempex, Roche, Salvat, Scynexis, Seachaid,
Shionogi, Tetraphase, The Medicines Co, Theravance, ThermoFisher,
VenatoRX, Vertex, Wockhardt, and Zavante. J. F. M. has received grants
from Astellas, Basilea, and Merck, has been a consultant to Astellas, Basilea,
and Merck, and has received speaker’s fees from Merck, United Medical,
and Gilead Sciences. All other authors report no potential conflicts.
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