Candida auris Infection in a Meningococcal Septicemia Survivor, Poland

Background Candida auris is an emerging pathogen that constitutes a serious global health threat. It is difficult to identify without specific approaches, and it can be misidentified with standard laboratory methods, what may lead to inappropriate management. Case Presentation We report, probably the first in Poland, C. auris isolation from blood cultures and wound swabs of a young male following meningococcal septicaemia, in February 2019. The patient had been previously hospitalized in the United Arab Emirates. The isolate was rapidly identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and therefore clinicians were promptly informed on the alert pathogen isolation. The targeted antifungal treatment was successful and infection control measures seemed effective. ITS-based identification and subsequent whole genome sequencing showed that the C. auris isolate belongs to South Asian lineage (clade I). Conclusions C. auris is able to cause outbreaks in healthcare settings. Therefore, it is important to quickly identify C. auris isolates in hospital settings so that healthcare facilities can take proper precautions to limit its spread.

quickly identify C. auris isolates in hospital settings so that healthcare facilities can take proper precautions to limit its spread.
Keywords Candida auris Á Candidaemia Á Infection prevention Á MALDI-TOF MS Á Poland Á Whole genome sequencing Background Candida auris has been widely known as an emerging multi-drug resistant fungal pathogen. It might be resistant to all available classes of antifungals [1][2][3][4]. This species tends to persist on a human host and on inanimate surfaces for months [5]. In contrast to other Candida species, C. auris spreads easily in health-care facilities causing sporadic cases and outbreaks [1][2][3][4][5].
Here we report the isolation of C. auris from a patient in Poland, the successful targeted antifungal treatment of an invasive infection, and the effective infection control procedures.

Case presentation
An eighteen years-old male was admitted in February 2019 to the Dr Antoni Jurasz University Hospital No. 1 in Bydgoszcz, Poland, to the Department of Transplantation and General Surgery, for a treatment of severe complications of a meningococcal septicaemia. The patient was transferred directly from the district hospital in Greater Poland, after two days of hospitalization and following three weeks of hospitalization in the United Arab Emirates (UAE), due to a meningococcal septicaemia.
At the admission, two blood samples and seven swabs from necrotic chronic wounds were collected. The aerobic blood culture became positive after two days of incubation (BD BACTEC TM FX system, Becton, Dickinson and Company, Sparks, MD, USA).
Microscopic examination revealed the presence of budding, non-pseudohyphae-forming, cylindrical shaped yeast cells (Fig. 1). An empiric treatment with an intravenous fluconazole was administered.

Identification of Candida auris
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the MALDI Biotyper system (Bruker Daltonics, Bremen,Germany) provided a confident species-level identification of the isolate (score 2.16). The clinicians were immediately informed on C. auris isolation.
Besides, multifocal C. auris colonization of necrotic chronic wounds on upper and lower limbs, and subsequently-stumps, was documented. A total of 20 C. auris isolates were cultured (Table 1).

Antifungal Susceptibility Testing
Antifungal susceptibility testing was performed by Etest method (bioMérieux, Marcy-l'É toile, France) according to the manufacturer's instructions. The   Table 1. The results interpretation was performed according to CDC guidelines (Centers for Disease Control and Prevention, CDC) [1].

Treatment
Therapy with micafungin 100 mg/day was initiated. Octenilin Ò and Octenisept Ò (Schülke & Mayer, Norderstedt, Germany) were used topically. Altogether, four positive blood cultures were obtained, all in aerobic conditions. Simultaneously incubated blood cultures in an anaerobic conditions were negative. The first negative blood culture in the follow-up investigation was noticed after seven days of treatment with micafungin. The treatment was continued for two weeks in total. The susceptibility of the isolates to micafungin was monitored, and they remained susceptible to the drug. However, a MIC value increase from 0.047 to 0.125 mg/L was observed.
In contrast to the eradication of C. auris from blood, a persistent colonization of necrotic chronic wounds of upper and lower limbs with C. auris was documented.

Molecular Characterization
Culture for DNA Isolation DNA was isolated from the 48 h culture of the clinical C. auris isolate (no. 5049-2019) and type C. auris DSMZ 21,092 strain with an application of ExtractMe DNA Yeast Kit (DNA Gdańsk, Gdańsk, Poland), performed according to manufacturer's instruction. The DNA samples concentration quality was checked spectrophotometrically and the isolation protocol efficiency was confirmed additionally electrophoretically in 1.5% agarose in TBE buffer (Bio-Rad, Feldkirchen, Germany). DNA quality control was performed by measuring the absorbance at 260/280 nm, the template concentration was determined using Qubit fluorimeter (Thermo Fisher Scientific, Waltham, Massachusetts, USA). DNA integrity was analyzed by 0.8% agarose gel electrophoresis.

ITS Region Sequencing
The ribosomal DNA ITS-region was amplified using primers ITS1 and ITS4 as previously described [6]. An amplification was performed using an Applied Biosystems 9700 Thermal Cycler according to cycling conditions described by Carolus et al. [6]. An amplification results were checked on a 1% agarose in TAE buffer. PCR product was cleaned using Amicon Ultra 100 kDa filters (Millipore, Burlington, Massachusetts, USA) and sequenced using BigDye TM Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) according to manufacturer's instructions. The sequencing products were electrophoresed on an ABI 3130 DNA analyzer using 36 cm capillaries and POP-7 polymer. The sequences were compared with these corresponding to a particular clade [6]. The type C. auris DSMZ 21,092 strain DNA sequence of the ITS locus was identical to the reference sequence of clade II. Clinical C. auris isolate was recognized as clade I or III, therefore whole genome sequencing (WGS) was performed to define the clade.
A genomic relationship of tested C. auris isolate (Candida-auris-PL1) reveals that it belongs to clade I. The reference strain for the phylogeny analysis was C. auris B8441, which belongs to clade I (NCBI Genbank ID: PEKT00000000.2). WGS was performed and the obtained data were analysed at the DNA Sequencing and Oligonucleotide Synthesis Laboratory, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences (IBB PAS).
WGS analysis of clinical C. auris isolates discovered four major clades: South Asian (clade I), East Asian (clade II), African (clade III), South American (clade IV), and Iranian (clade V), which means simultaneous emergence of multidrug-resistant C. auris on different continents [12,14,15]. Simultaneously, Tsay et al. [16] revealed the transmission of C. auris isolates in health care facilities within several United States.
C. auris strains firstly appeared in Europe in 2015 [5]. The outbreak involved over 50 cases in a London hospital (2015-2016), and the ward closure was implemented to control outbreak. Thereafter, sporadic cases and outbreaks in Europe have been reported from: Austria, Belgium, France, Germany, Greece, Italy, the Netherlands, Norway, Spain, Switzerland, and as we here reported, in Poland [3,4,[17][18][19][20].
To our knowledge this case is the first report of C. auris from Poland. Applied molecular methods confirmed that the isolate belongs to clade I, also known as South Asian. It is presumed that C. auris infection and colonization was ongoing when the patient was admitted to our hospital, but the Candida sp. was not isolated or identified to the species-level in the United Arab Emirates (UAE). A sporadic case of C. auris isolation in the UAE has been previously reported [21]. Widely used phenotypic methods mostly fail to identify this novel Candida species. At present, mass spectrometry (e.g. MALDI Biotyper, Bruker, and Vitek MS, bioMérieux) and DNA sequencing of the ITS and/or D1/D2 ribosomal DNA regions reliably identify C. auris [1,22]. Still there is inability to identify C. auris accurately in a large number of countries.
Fortunately, we were able to identify the pathogen rapidly and reliably, and inform the clinician promptly. Hence, contact precautions were introduced immediately to prevent this pathogen transmission.
The actual problem of C. auris is a lack of standards for disinfection procedures effective against C. auris. The protocols applied for infection control in our healthcare facility seem effective as until time of publication, any other C. auris isolation ensued.
Likewise the majority of C. auris strains [1,3], our isolate was resistant to fluconazole, therefore the treatment with echinocandin was required. We have monitored the antifungal susceptibility of the isolates during therapy, and they remained susceptible to echinocandins until the end of the treatment.
C. auris tends to persists in patients, especially on skin of groins and axillae, in ear canals, and in nostrils [5]. We have also observed persistent colonization of the chronic wounds on limbs, over a month after a successful treatment of candidaemia.
Our case proved that careful and precise Candida spp. identification to the species level is of great importance. There is a high need of a reliable and rapid identification of C. auris in all medical microbiology laboratories using new technologies approved for microbiological diagnostic. This is the basic tool for an effective prevention of infection and control measures.
In conclusion, our report confirms the ongoing introduction of C. auris into European hospitals and the role of a reliable and rapid C. auris identification in prevention of further transmission and healthcareassociated outbreaks. Difficulties in an identification may delay early pathogen detection, increasing the risk for C. auris transmission. A cooperation of microbiologists, clinicians, nurses and epidemiologists is particularly important in this case. There is a constant need to evolve standards for infection prevention and control measures effective against C. auris.
Acknowledgements The authors are grateful to Jan Gawor M.Sc. from DNA Sequencing and Oligonucleotide Synthesis Laboratory, IBB PAS, for performing WGS and phylogenetic analysis.
Authors' Contributions MP: analysis and interpretation of patient's cultures; isolation and identification of C. auris; performing and interpretation of susceptibility tests; integration of data submitted by all contributors; drafting and revising the manuscript; PZW: analysis and interpretation of patient's cultures; isolation and identification of C. auris; prompt notification of C. auris to the clinicians and infection control team; performing and interpretation of susceptibility tests; contribution to critical revision of the manuscript; ZW: patient treatment; administration of antimicrobial therapy; revising the clinical aspects of the manuscript; AD: head of infection control team and antimicrobial stewardship consultant; designing of antimicrobial treatment plan for the patient, epidemiological analysis of patient; drafting and revising the manuscript; EGK: analysis and interpretation of integrated data; critical revision of the manuscript; TB: DNA isolation, sequencing and analysis, critical revision of the manuscript; MW: DNA sequencing and analysis, critical revision of the manuscript. All authors have read and approved the manuscript. Ethics Approval and Consent to Participate Not applicable.
Consent for Publication Written informed consent for publication of this case report was obtained from the patient.
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