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ORIGINAL UNEDITED MANUSCRIPT
Candida auris: Outbreak, surveillance and
epidemiological monitoring in Northern Greece
Αikaterina Poulopoulou1, Anna Sidiropoulou1, Theopisti Sarmourli1, Evaggelia Zachrou1,
Chrysi Michailidou2, Charalampos Zarras2, Eleni Vagdatli2, Eleni Massa3, Eleni Mouloudi3,
Athina Pyrpasopoulou4, Georgios Meletis5, Efthymia Protonotariou5, Lemonia Skoura5,
Simeon Metallidis6, Theodoros Karampatakis7, Eleni Katsifa7, Anna Nikopoulou8,
Alexandra Louka9, Artemisia Rizou9, Kostoula Arvaniti10, Vassili Kouvelis11, Andrew
Borman12, Emmanuel Roilides4, Timoleon-Achilleas Vyzantiadis1
1Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Greece
2Department of Biopathology, Hippokratio General Hospital, Thessaloniki, Greece
3ICU, Hippokratio General Hospital, Thessaloniki, Greece
4Infectious Diseases Unit, Hippokratio General Hospital, Thessaloniki, Greece
5Department of Microbiology, AHEPA University Hospital, Medical School, Aristotle University of
Thessaloniki, Greece
6First Department of Internal Medicine, Infectious Diseases Division, AHEPA University Hospital,
Medical School, Aristotle University of Thessaloniki, Greece
7Department of Microbiology, G. Papanikolaou General Hospital, Thessaloniki, Greece
8Infectious Disease Unit, G. Papanikolaou General Hospital, Thessaloniki, Greece
9Department of Microbiology, Mamatsio General Hospital, Kozani, Greece
10ICU, Papageorgiou General Hospital, Thessaloniki, Greece.
11Section of Genetics & Biotechnology, Department of Biology, National & Kapodistrian University
of Athens, Greece
12Mycology Reference Laboratory, UK Health Security Agency, Southmead Hospital, Bristol, UK
Authors names and degrees:
Αikaterina Poulopoulou (Ms), Anna Sidiropoulou (Ms), Theopisti Sarmourli (Dr), Evaggelia
Zachrou (Ms), Chrysi Michailidou (Dr), Charalampos Zarras (Dr, PhD), Eleni Vagdatli (Dr,
PhD), Eleni Massa (Dr), Eleni Mouloudi (Dr, PhD), Athina Pyrpasopoulou (Dr, PhD),
Georgios Meletis (Dr, PhD), Efthymia Protonotariou (Dr, Associate Professor), Lemonia
Skoura (Dr, Professor), Simeon Metallidis (Dr, Professor), Theodoros Karampatakis (Dr,
PhD), Eleni Katsifa (Dr), Anna Nikopoulou (Dr, PhD), Alexandra Louka (Dr, MSc), Artemisia
Rizou (Dr), Kostoula Arvaniti (Dr, PhD), Vassili Kouvelis (Dr, Associate Professor) Andrew
Borman (Professor), Emmanuel Roilides (Dr, Professor), Timoleon-Achilleas Vyzantiadis
(Dr, Professor)
Corresponding author:
Timoleon-Achilleas Vyzantiadis, MD, PhD
Professor of Medical Biopathology-Microbiology
Department of Microbiology
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ORIGINAL UNEDITED MANUSCRIPT
Laboratory of Medical Mycology
Medical School
Aristotle University of Thessaloniki
Thessaloniki 54124
Greece
email: avyz@med.auth.gr
Key words: Candida auris, outbreak, epidemiology, monitoring, Greece
Conflict of interest: The authors declare no conflict of interest.
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ORIGINAL UNEDITED MANUSCRIPT
Abstract
Candida auris is an emerging fungal pathogen associated with multi-drug resistance rates
and widespread outbreaks in hospitals and health care units worldwide. Sequencing
studies have revealed that different clonal lineages of the fungus seem to be prevalent
among distinct geographical sites. The first case of C. auris in Northern Greece was
reported in Thessaloniki in October 2022, almost two years after the first isolation in
Greece (Athens 2019). The Mycology Laboratory of the Medical School of Aristotle
University of Thessaloniki stands as the reference laboratory for fungal diseases in
Northern Greece and a meticulous search for the yeast, in plenty of suspicious samples,
has been run since 2019 in the Lab as well as a retrospective analysis of all its yeasts’
collection, back to 2008, with negative results for the presence of C. auris. Here, are
presented the findings concerning the outbreak and surveillance of C. auris in Northern
Greece, mainly the region of Thessaloniki and the broader area of Macedonia, from
October 2022 until August 2023. The isolates from Northern Greece continue to fall in
Clade I and present with an almost equal and stable sensitivity profile until now.
Lay Abstract
The study concerns the outbreak of Candida auris in Northern Greece since October 2022
and the effort for surveillance and epidemiological monitoring. All isolates continue to fall
in Clade I and present with an almost equal and stable sensitivity profile till now.
Introduction
Candida auris has evolved to a genuine threat to public health, particularly within
nosocomial settings, due to its ability to cause invasive infections and lead even to fatal life
events.1 The emergence of C. auris is connected to its environmental resilience, horizontal
transmission, multi-drug resistant profile and finally its recurring misidentification.2C.
auris was first isolated in Japan in 2009 from the external ear canal of a hospitalised
patient.3 Subsequently, in South Korea, C. auris was isolated from 15 patients with chronic
otitis media.4 However, the earliest case of C. auris was found retrospectively from an
initially misidentified sample that was collected from a patient with fungaemia in South
Korea in 1996.5 In the last few years, C. auris infections have been reported across several
countries and regions all around the world.6 In Greece, C. auris was first isolated in 2019 in
Athens from a sputum culture of a cystic fibrosis patient.7
Regarding the evolution of C. auris, it belongs to the Clavispora clade of
Metschnikowiaceae family of the order Saccharomycetales.8 Whole genome sequencing
(WGS) studies have identified five genetically diverse clades on C. auris phylogenetic tree
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ORIGINAL UNEDITED MANUSCRIPT
(South Asian Clade I, East Asian Clade II, South African Clade III, South American/Israeli
Clade IV and Iranian Clade V).9,10 Recently a sixth clade (Clade VI) has been identified in
Singapore and Bangladesh, revealing a strong association with strains of Clade IV in South
America.11 These clades of C. auris are genetically highly related (98.7% average pairwise
nucleotide identity) and all of them are genetically distanced of other resembling Candida
species, such as C. haemulonii, C. duobushaemulonii and C. psuedohaemulonii.12
Candida auris infections mainly occur in critically ill hospitalised patients. Apart from the
bloodstream infections, there have also been isolations from the respiratory tract, the
urinary tract, the skin and wounds, even the central nervous system (CNS).13,14 On the
other hand, C. auris easily colonises the skin and many body sites of patients in health care
units.15 As C. auris is also able to survive outside of the human body, the fungal
transmission can occur via hospital surfaces, contaminated medical equipment and finally
via the hands of medical staff.16,17,18,19,20
The data from the European Centre for Disease Prevention and Control (ECDC) show that
there are continuously increasing numbers of either colonisation or invasive cases of C.
auris in Europe, together with large and prolonged nosocomial outbreaks. Why some
cases caused large outbreaks and others remained sporadic is probably related to the low
numbers of isolates that might reach the mycology reference laboratories in reality.
Additionally, the lack of adequate information for the surrounding contact screening of
these isolates in order to exclude further transmission could be another cause.21
C. auris has demonstrated high rates of drug resistance. A great percentage of C. auris
isolates are reported resistant to fluconazole (in Clade 1, this has been reported in more
than 90% of isolates), while elevated minimum inhibitory concentrations (MICs) to
amphotericin B have been reported several times, and resistance to echinocandins is
emerging in some countries.9,22 It is also true that there is a worldwide increased
detection of multidrug-resistant (MDR) or even pan drug-resistant (PDR) strains of C.
auris.23 The resistance levels vary between the clades, while several mutations seem to be
associated by clade.9,22,24
All of the above indicate the need for monitoring its emergence in a coordinated manner
and further highlights the need for adequate mycology reference capacity. In the UK, the
early detection of outbreaks and early isolation enhanced the infection control measures
and the screening was able to control the spread of the fungus.25 Good knowledge of local
epidemiology and control of Candida auris could delay its establishment in the relevant
healthcare facilities and finally have a broader nationwide benefit by reducing the
healthcare associated infections.26
In this context, the main target of this work (that still continues in full scale) was to
monitor in a well organised and centralised procedure the spread of C. auris in our
broader geographical region by providing full laboratory adequacy in its early isolation
and full identification, as well its phylogenetic categorisation and screening of its
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sensitivity level to the commonly used antifungals. All this data will undoubtedly be a
useful tool in the general antifungal stewardship of C. auris (when needed) and the
implication of appropriate control measures of its spread.
Methods
Ninety isolates of C. auris were referred or were isolated directly from culture specimens
that were sent to the Mycology Laboratory of the Medical School of Aristotle University of
Thessaloniki. The Lab serves as the reference laboratory for fungal diseases in Northern
Greece and the isolates were mainly from eight tertiary regional hospitals, from 89
patients and 1 oximeter (Table 1).
Most of the referrals concerned yeasts’ isolations with low score of identification or no
identification score, misidentification or even unusual identification at the automated
systems of the hospitals, isolates with unusual colors or colors similar to the expected
(depending on the several chromogenic media) for C. auris at the chromogenic media and
isolates with higher Minimum Inhibitory Concentrations (MICs) at fluconazole. Even in the
case that a MALDI-TOF existed, a low score value or an inconclusive result was again a
reason for referral. Also, a smaller part of the isolations of C. auris occurred at the primary
culture of the biological specimen directly in the Ref Lab.
The first isolate was reported in Thessaloniki in October 2022 and the procedure is still
ongoing as part of a continuous monitoring and surveillance process. This study includes
the isolates reported until early August 2023. Since then, another 50 additional isolates
were received from at least 10 different hospitals, till the end of 2023. Identification and
confirmation at the Ref Lab involved phenotypical methods such as microscopy, growth at
42°C and use of chromogenic media (CHROMagarTM Candida Plus, CHROMagar, Paris,
France), as well as an in-house evaluated PCR27 and MALDI-TOF MS (MALDI Biotyper,
Bruker Daltonik GmbH, Bremen Germany) or / and in many cases molecular confirmation
by a combination of rDNA gene amplification and sequencing targeting of the ITS1 rRNA
and / or 28S regions. The latter, were cases randomly selected when preparing for
phylogeny, when there was a case of a “new” hospital in the C. auris repertoire or when
there were indices for higher MICs. At least one third (32) of the described ninety isolates
were gradually sequenced by targeting one of the two mentioned regions, and at least half
(16) of them (as representative isolates, according to the different hospitals), were
sequenced for both regions (ITS1 and 28S).
Hospitals included in the study have different methodologies for species detection and
identification, namely numerous surveillance and diagnostic cultures, inoculation onto
diverse chromogenic media, the use of automated systems like the Biomerieux Vitek-2 or
the BD Phoenix. During the study period, three of those hospitals (regional Hospitals)
acquired equipment based on MALDI-TOF MS technology, and therefore, this specific
methodology was included in the local identification procedure.
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Minimum Inhibitory Concentrations (MICs) for various antifungal drugs were measured
using both commercial and reference methods.
The methods provided complementary, but also in some cases confirmatory results (in
cases of higher MICs) to every day’s routine work and the continuous effort to provide in
short time a comprehensive report to the referring Departments. Many times, as this was
a prospective and demanding continuous daily work, the methodological use was a matter
of availability in reagents, time and personnel.
The methods were the ATB-Fungus ATB™ Fungus 3 (bioMérieux SA, Marcy l’ Etoile,
France) for amphotericin B, flucytosine, fluconazole, itraconazole and voriconazole, the
MIC Test Strips (Liofilchem srl, Roseto degli Abruzzi, Italy) for posaconazole, voriconazole,
amphotericin B, isavuconazole, micafungin, caspofungin and anidulafungin, and an “in
house” prepared version of the EUCAST reference method in the context that the
concentrations of the tested antifungals (flucytosine, amphotericin B, fluconazole,
itraconazole and voriconazole) were prepared in the same range as them of the ATB
Fungus. Also, a separate panel was prepared according to the EUCAST method (range
0,0312-16 μg/mL) for the possible need of further confirmation of the MICs of the three
echinocandins.28 However, none of the studied isolates was considered resistant to the
echinocandins in order to need further confirmation.
MICs evaluation was done according to the tentative breakpoints that have been
suggested by the US Centers for Disease Control and Prevention (CDC), combined to
others by expert opinions and comparative studies.12,29,30
Most of the results of identification and sensitivity testing were reported to the relevant
Hospitals' Departments in maximum during the third day after the arrival of the yeast
isolate in the Ref Lab.
Fungal genomic DNA was extracted from all C. auris isolates by heating at 95oC under
buffered conditions, followed by strong agitation and centrifugation.31,32 Among the
strains that were sequenced for both, ITS1 and 28S (D1-D2) ribosomal regions, ten
representative strains (according to the referring hospital and at least one strain per
Hospital as following: M739-22, M860-22, M14-23, M57-23, M71-23, M76-23 A, M88-23,
M114-23, M136-23 B, M152-23) were used for multilocus phylogenetic analysis. The
relevant isolates’ sequences of the fungal ribosomal DNA were deposited at GenBank
under the accession numbers PP177375, PP178700-PP178708 for the ITS1 region and
PP317006-PP317015 for the 28S region, respectively.
The studied sequences were aligned with reference sequences of C. auris according to the
main four clades and the related geographic origins, as sourced from GenBank. Alignment
was performed using ClustalW.33 As an outgroup, a sequence of the closely related
Candida haemulonii was used. Phylogenetic analysis utilised both the neighbor-joining
(NJ) and Bayesian inference (BI) techniques, conducted through MEGA X34, PAUP35, and
MrBayes (ver. 3)36. These analyses followed the methodologies and parameters outlined
in prior research for constructing all phylogenetic trees.37,38 The Kimura-2 parameter was
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ORIGINAL UNEDITED MANUSCRIPT
chosen for NJ analyses; whereas the jModelTest program (ver. 2)39 determined the
optimal model for BI analyses for the specific matrix. Statistical support for nodes was
assessed using 500 bootstrap replicates.
Results
The isolates (45 from ICUs, 38 from Internal Medicine and Subspecialties’ Departments, 7
from Surgical Departments) were isolated from 89 hospitalised patients (57 men-32
women, mean age: 66.77±16.68 years, ranging 18-92) and one oximeter that was not
connected directly to a specific patient. According to the available data, 40 isolates
concerned colonisation and 22 deeper sites (among them, blood and urine, 3/14). There
were 27 recorded deaths but only one was directly attributed to C. auris. According to the
methodology described above in the methods section, all isolates were resistant to
fluconazole (geometric mean: 97.76 μg/mL, 32-256, modal 128) with a sporadic tendency
for higher MICs for voriconazole (gm: 1.17 μg/mL, 0.125-4, modal: 0.5) and sensitive to all
other drugs such as amphotericin-B, 5-flucytosine, isavuconazole, itraconazole,
posaconazole, and echinocandins (Table 2).
Phylogenetic analyses of the combined 28S (D1/D2) and ITS1 datasets revealed that the
current isolates from Northern Greece are classified (Figure 1) within the South Asian
lineage (Clade I), similarly to other strains from Greece until today.7,40 Monitoring is
ongoing, with new strains being added daily to the Mycology Lab’s collection but with no
differentiation of the total image until now (same sensitivity profile and phylogeny).
Discussion / Conclusion
Candida auris presents increased ability for colonisation and horizontal spreading and
tends to affect seriously ill, more vulnerable hospitalised patients. It can be a direct threat
for clinical prognosis and normal workflow in the health system, and influences the health
care infrastructure and finance.41,42
It was evident also during this study that the challenges to be faced both by the Hospitals’
Departments as by the Ref Lab in terms of application of adequate measures and protocols
or the availability of reagents, personnel, time and finance were present, mostly due to the
need to react fast and efficiently against a severe outbreak with numerous cases. It was
proved once more that the constant need for a well prepared and adequately supported
public health system with several consecutive layers of preparedness and responsiveness
will be always a necessity.
The first isolate was reported in Thessaloniki in October 2022 almost two years after the
first isolation in Greece (Athens 2019)7 and here are presented the findings concerning
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ORIGINAL UNEDITED MANUSCRIPT
the outbreak and surveillance of C. auris in Northern Greece, mainly the region of
Thessaloniki and the broader area of Macedonia, from October 2022 until August 2023.
Since then, another 50 additional isolates were received from at least 10 different
hospitals, until the end of 2023, suggesting that the outbreak should not be perceived as
local but of broader extent in Northern Greece.
The meticulous search for the yeast, that has been run since 2019 in the Lab by identifying
all (even a single colony) yeast colonies from the cultures of all biological samples that
were referred to the Lab, as well as the retrospective analysis of all its yeasts’ collection,
back to 2008, showed negative results for the presence of C. auris until October 2022.
Although it seems for the moment that the continuing spread of C. auirs in our region is
under relative control and that there are still therapeutic alternatives, nothing is certain
for the future.
Τhe phylogenetic analysis of the combined 28S (D1/ D2) and ITS1 ribosomal regions
revealed that the current isolates from Northern Greece are classified within the South
Asian lineage (Clade I), similarly to other strains from Greece.7,40 As this phylogenetic
analysis was based on a rather small number of representative isolates, more isolates will
be added to enhance the data robustness, as the outbreak progress.
As elsewhere, C. auris isolates from our region are highly resistant to fluconazole.43
Isolates of Clade I exhibit resistance to fluconazole and variable resistance to
amphotericin B.44 However, the latter is not still the case for our region, neither did we
found any in vitro resistant strain in echinocandins.
Unfortunately, clinical breakpoints for minimum inhibitory concentration (MIC) for C.
auris have not been established. However, tentative breakpoints that have been suggested
by the US CDC, combined to others by expert opinions and comparative studies, can
provide a quite safe approach for the in vitro resistance level of the yeast.12,29,30
Echinocandins seem to be for now the treatment of choice.29 However, its constant and
broad use could endanger the development of resistance to them.9
Sequencing of genetic loci as rRNA D1/ D2 and ITS domains is useful in the identification
of C. auris. However, it is not routinely and easily used and is unlikely to be available
outside reference laboratories.42 Although its ability to differentiate between geographic
clades has been demonstrated,45 its ability to discriminate between strains is possibly
limited.46 For this reason, several other molecular techniques as amplified fragment length
polymorphism (AFLP) analysis, pulsed-field gel electrophoresis (PFGE), M13 DNA
fingerprinting, together with the sequencing of genetic loci, have been proposed for the
typing of C. auris isolates.1,42
A comparative analysis of ITS sequencing, AFLP genotyping and microsatellite typing
showed that although microsatellite and ITS typing had technical reproducibility and
agreement, the ITS sequencing cannot be reliably used to split the C. auris into geographic
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ORIGINAL UNEDITED MANUSCRIPT
clades.47 Microsatellite typing offered better resolution and a good concordance with
whole genome sequencing (WGS) analysis together with lower costs compared to the
WGS,48 while the background information about the phylogeographic structure of C. auris
was studied and provided earlier using WGS and ITS sequencing.
Like in our study, the combined phylogenetic analysis of both D1/D2 and ITS1 regions has
been proved previously as a useful tool in studying the several clonal lineages of C. auris
stains. However, since there are low levels of genetic variation between isolates from a
specific clonal origin, WGS will probably be a more appropriate tool than multilocus
sequence typing (MLST) in order to further delineate between strains as it was also
showed during an additional preliminary analysis in our Laboratory by the use of
nanopore technology45,49
The use of phylogeny tools and genomic surveillance holds promise in strengthening C.
auris surveillance networks in the future29 while laboratories engaged in surveillance
should be able to compare analyses with diverse national and international collaborators,
ensuring that findings are mutually reliable and comprehensible. These data are even
more important as there are specific susceptibility patterns associated with different
clades. 9, 12, 22, 25, 30, 50
In total, well organised surveillance approaches and continuous monitoring are constantly
needed in order to keep a high level of preparedness and efficient response.
Disclosure of Conflict of Interest: The authors declare no conflict of interest.
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Table 1. Τype and origin of the ninety isolates
Gender
F
M
n
umber
32
57
age (mean± SD)
67.96±14.67
64.44±17.82
Colonisation
Candidaemia
Other deeper sites
(among them, urine)
number of isolates*
40
3
19
(14)
Hospitalisation Unit
number of isolates
ICU
45**
Surgery
7
Internal Medicine
34
Pulmonology
1
Haematology/Transplantation
2
Cardiology
1
*according to the provided clinical data
**one isolate is from an oximeter
Table 2. MIC patterns of Candida auris isolates
5-FC
AP-B FL ITRA VOR POS AND MYC ISA
Range
4-32 0.5-1.0 32-256 0.125-0.5 0.125-4 0.002-0.5 0.002-2 0.032-0.125 0.008-1.5
GM 4.09 0.79 97.76 0.19 1.17 0.08 0.08 0.06 0.11
Modal
4 1 128 0.125 0.5 0.064 0.125 0.064 0.094
*GM: geometric mean
5-FC: 5-Flucystosine, AP-B: Amphotericin-B, FL: Fluconazole, VOR: Voriconazole, POS:
Posaconazole, AND: Anidulafungin, MYC: Micafungin, ISA: Isavuconazole
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ORIGINAL UNEDITED MANUSCRIPT
Figure 1. Phylogenetic tree corresponding to the merged D1/D2 and ITS1 data sets,
produced by BI analyses.
Alt-text. Graphical phylogenetic depiction of the separate clades, according to the
combined D1/D2 and ITS1 ribosomal regions of the studied strains of C. auris.
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