Candida auris Updates: Outbreak Evaluation through Molecular Assays and Antifungal Stewardship—A Narrative Review

Candida auris was reported by the WHO as second to Cryptococcus neoformans, in the list of nineteen fungal priority pathogens, along with two species with a new nomenclature, Nakaseomyces glabrata (Candida glabrata) and Pichia kudriavzevii (Candida krusei). This novel classification was based on antifungal resistance, the number of deaths, evidence-based treatment, access to diagnostics, annual incidence, and complications and sequelae. We assessed which molecular assays have been used to diagnose Candida auris outbreaks in the last five years. Using “Candida auris; outbreak; molecular detection” as keywords, our search in PubMed revealed 32 results, from which we selected 23 original papers published in 2019–2024. The analyzed studies revealed that the detection methods were very different: from the VITEK® 2 System to MALDI TOF (Matrix-Assisted Laser Desorption Ionization–Time of Flight), NGS (Next-Generation Sequencing), WGS (Whole Genome Sequencing), and commercially available real-time PCR (Polymerase Chain Reaction) assays. Moreover, we identified studies that detected antifungal resistance genes (e.g., FKS for echinocandins and ERG11 for azoles). The analyzed outbreaks were from all continents, which confirms the capability of this yeast to spread between humans and to contaminate the environment. It is important that real-time PCR assays were developed for accurate and affordable detection by all laboratories, including the detection of antifungal resistance genes. This will allow the fast and efficient implementation of stewardship programs in hospitals.


Introduction
In 2022, the World Health Organization (WHO) Antimicrobial Resistance Division released a report in which the rising invasive fungal diseases were compared to drugresistant bacterial infections.Both bacteria and fungi represent important medical issues, especially fungal infections, which are challenged by limited access to quality diagnostics and treatment.
The pathogens included were classified and then categorized into three priority groups (critical, high, and medium).The critical group includes Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, and Candida albicans.The WHO endorses including fungal diseases and priority pathogens in medical (clinical) and public health training programs and encourages optimizing the mycology diagnostic capacity to manage fungal infections and to perform surveillance [1].Although taxonomical changes may prove difficult to adapt to, the recent fungal reclassification is another step in underlining the importance of fungal resistance and the possible emergence of novel pathogens.Recently, a change in the nomenclature of fungi has been underway, reclassifying fungal species into new genera based on antifungal resistance and thermotolerance [2].The WHO priority fungi list mentions two such reclassified species: Nakaseomyces glabrata (Candida glabrata) and Pichia kudriavzevii (Candida krusei) in the high and medium priority groups, respectively [1].
Candida auris, a critical priority fungal species according to the WHO, emerged as a yeast that is multidrug-resistant in the past decade.This species inhabits the surface of the human skin and hospital settings, leading to the occurrence of healthcare-associated epidemics and invasive candidiasis.The initial genetic studies identified four primary clades, which were designated after their corresponding geographical locations: South Asia, East Asia, Africa, and South America.These clades are referred to as clade I, clade II, clade III, and clade IV, respectively, and are differentiated by at least ten thousand singlenucleotide polymorphisms (SNPs) between any two clades.In 2022, a fifth clade, from Iran, was confirmed by whole-genome sequencing [3].
The first case of Candida auris was isolated in 2009 from a sample of ear discharge from the external ear canal of a 70-year-old Japanese woman, as reported by Satoh K et al. [4].The authors proposed this new species based on a single isolate, after the comprehensive description of the fungus (Latin diagnosis and description of Candida auris).The phylogenetic tree of JCM15448T in the 26S D1/D2 domain (AB375773) and ITS region (AB375772) sequences were also presented in that paper [4].
The first seven cases of C. auris identified in the USA were presented by the CDC (Centers for Disease Control and Prevention) in 2016, by Vallabhaneni S et al., which included the analysis of five blood samples, one urine sample, and one ear pus collection [5].The authors mentioned the importance of using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF) or sequencing the D1-D2 region of the 28S ribosomal DNA, as biochemical assays, for example, VITEK 2, seem not to be sensitive enough for the accurate detection of this fungal species.By whole-genome sequencing (WGS), the authors detected high similarities between those seven strains, and then concluded that C. auris could be considered an emerging pathogenic fungus, multidrugresistant, with some strains having elevated minimum inhibitory concentrations (MICs) to drugs belonging to all major classes of antifungal medications.The authors also reported that this fungus has the capability of spreading within healthcare settings [5].
Two years later, Chow NA et al. published in 'The Lancet' the results of a WGS-molecular epidemiological survey of C. auris in ten US states.The authors found that all C. auris isolates isolated from the patients assessed in the USA belonged to one of the four known clades (African, South American, East Asia, and South Asia), suggesting several introductions of C. auris into the USA.Molecular epidemiology has provided key insights into the origin and transmission dynamics of C. auris in US healthcare facilities.The authors underlined that measures are needed in preparing for outbreaks and identifying this new multidrug-resistant yeast in order to contain its spread and transmission within the USA [6].
Another very recent 'Lancet' paper from 2023 published the characteristics of the five cases of coinfection from a southern Nevada dual-clade outbreak of Candida auris.The coinfections were isolated from different samples (two from the axilla, blood, and urine; two from the axilla and urine; and one from urine and wound sample).For the strains isolated from blood and urine samples, the capsofungin MIC value was determined.The available phenotypic susceptibility data showed that, in the isolates with a mutation in the FKS1 gene, the MIC values for caspofungin were elevated when compared to wild-type isolates [7].
The aim of this review was to summarize the recent findings regarding Candida auris's molecular diagnosis in worldwide outbreaks registered in the last five years.To date, there is no FDA-approved assay for this fungus, and therefore, hospital microbiology laboratories should be properly informed, from a scientific and logistic point of view, regarding which assay is most suitable to be implemented for the fast and accurate diagnosis of C. auris.

Worldwide Guidelines Elaborated to Control C. auris Outbreaks
Many countries have elaborated new protocols for the early detection, treatment, and prevention of C. auris infections.Boyce JM. et al., from the USA, stated in a recent paper that the potential infection risk of this newly described fungus is on the same level as SARS-CoV-2 and mpox.The authors highlighted the importance of utilizing efficient alcohol-based hand sanitizers, as there is still research into the factors impacting hand hygiene practices [8].A collaborative study that included almost all the countries from Europe published the initiative of The European Confederation of Medical Mycology (ECMM) with the intention to share their broad expertise on difficult-to-treat invasive fungal infections, and the advice will follow recent guidelines, including EQUAL (European QUALity (EQUAL) score).This Mycology Confederation wants to improve treatment outcomes and increase survival rates for difficult-to-treat invasive fungal infections [9].One year prior to this study, the same team of Koehler P. et al. developed the international Candida Registry (FungiScope™ CandiReg) to allow contemporary multinational surveillance.The registry was thought to be necessary, given the previously reported outbreaks of Candida auris and the potential of this pathogen to produce difficult-to-treat infections.The European Confederation of Medical Mycology includes 28 countries, and with the release of this online registry, it intends to promote international collaboration for an optimal management of Candida infections [10].In a comparative study for the period of 2013-2017 in a hospital from Johannesburg, South Africa, the authors highlighted the emergence of Candida auris, whose incidence increased from zero in 2013 to 11% in 2017.The researchers suggested that empirical antimicrobial therapy should be guided by an intensive care unit-specific antibiogram, and empirical antifungal therapy with amphotericin B or micafungin is adequate for patients that are at a high risk of invasive Candida infections [11].Ong CW et al. published in 2019 a review about the position of the Australasian Society for Infectious Diseases regarding the most suitable assay for Candida auris identification, MALDI TOF or sequencing, and stated that phenotypic methods could lead to misidentification.The Australasian researchers recommended echinocandins as first-line therapy for infection in adults and children ≥2 months of age.For neonates and infants <2 months of age, they recommended amphotericin B deoxycholate.Also, they suggested that composite swabs of the axilla and groin should be collected for screening patients for C. auris [12].Another guideline was elaborated by researchers from South Africa, which encountered 100 South African hospitals with cases of infections caused by Candida auris.Govender NP et al. released 18 practical recommendations, including the statement that the reference identification method should be molecular identification and the standardized broth microdilution (BMD) method should be used as a reference method for antifungal susceptibility testing.This guideline offers information regarding antifungal stewardship (e.g., diagnosis, stewardship, duration of therapy for sepsis, and antifungal consumption) [13].
All the above guidelines, produced by researchers from all continents, support the need for the immediate notification of C. auris isolation to clinical and infection control teams to prevent internal and inter-center transmission and to ensure the proper surveillance and prevention of infection of patients who are already colonized and can develop an infection.

Molecular Assays Used for the Active Surveillance of C. auris Outbreaks
In order to select the papers that would be included in this narrative review, we analyzed the molecular assays performed by different authors to assess Candida auris outbreaks in the last 5 years (2019-2024).Our search was initiated on 12 February 2024, and we searched the most prominent scientific databases, Web of Science-Clarivate and PubMed.For this process, we used the following keywords "Candida auris; outbreak; molecular detection".Thus, 68 results were revealed for the last 5 years.A total of 6 papers were excluded as their topic and scientific relevance did not fit the scope of our review, and thus, 62 papers were included in the present narrative review.
From these 62 articles, only 25 represented original research papers .
In Table 1, we include 34 papers, and it can be noticed the synthetic approach on the molecular detection of Candida auris in recent worldwide outbreaks (Table 1).We can observe that outbreaks with these new fungi have been documented worldwide: Europe (Spain, The Netherlands, and UK), Asia (Singapore, Japan, Iran, Turkey, China, Kuwait, and Lebanon), Africa (South Africa), USA, South America (Brazil), and Central America (Honduras).The most numerous studies for the analyzed period were from the USA (5/23), followed by Spain (3/23).The assays used were very diverse, from PCR with pan-fungal primers to MALDI-TOF MS, WGS of the microorganism, commercially available Eazyplex ® Candida auris kit, AmplexDiagnostics GmbH, Buseck, Germany, and a genosensor for C. auris.In total, these 23 analyzed papers tested 2770 clinical yeast samples for C. auris.One of the authors' study directions was to detect the new fungus directly from pathological samples, before previous DNA extraction, to improve the speed of the detection process.Another direction was to develop fully optimized assays, with high sensitivity and specificity, suitable for being commercially available.A very interesting and clinically useful approach was to detect, by real-time PCR or WGS, the genes that are known to be responsible for antifungal resistance: Y132F in ERG11, as the most widespread mutation associated with azole resistance, and S639P in FKS1 for echinocandin resistance.For antifungal susceptibility testing, the authors used different assays, such as the Vitek ® 2 System (Biomerieux, Denver, CO, USA) or Micronaut (Bruker, Billerica, MA, USA).The heterogeneity of the molecular assays used to detect C. auris can easily be observed, with different characteristics that are listed in Table 1.Of course, it would be ideal for healthcare providers to use clinically validated tests, recommended by official organizations in the field.In our analysis, we identified 25 papers, of which 6 used WGS + MALDI-TOF, 3 used MALDI-TOF + PCR/CULTURE, 11 used PCR, and 5 used other methods for C. auris detection.
The most comprehensive assay is WGS, of course, being a method that offers detailed genetic information, but has the disadvantage that it requires special and expensive laboratory equipment to be performed.MALDI-TOF is a very fast and accurate identification assay, and it needs to be followed by antifungal susceptibility testing.Real-time PCR allows for fast detection, and it was the most frequently used assay, probably because many laboratories, after the emergence of the SARS-CoV-2 pandemic, acquired the necessary equipment.More than simple detection, real-time PCR, which is able to detect antifungal resistance genes, seems to be even more suitable for fast diagnosis during Candida auris outbreaks.

Candida auris: Multidisciplinary Involvement
In the outbreak cases that we analyzed in this paper, there were diverse samples originating from ear aspirates, skin, blood samples, mucous membranes, and surfaces samples.We wanted to understand to which extent C. auris can be involved in other types of infections.C. auris was identified in a low-birthweight (800 g), preterm neonate in Italy in 2021.The authors recommended antifungal prophylaxis in low-birth-weight, preterm neonates with micafungin to avoid an unfavorable evolution [39].In a review by Sokou R et al. from 2024, 24 studies in neonates that were positive for this yeast were identified.The authors underlined the need of understanding Candida auris, whose pathogenesis is very important for developing effective strategies to control and prevent neonatal infections caused by this pathogen [40].
Interestingly, related to C. auris pathogenesis is the paper of Vila T et al. in the field of dental medicine.The authors used murine models of infection to comparatively evaluate the host niche-specific pathogenic potential of C. auris, and they found that C. auris adheres more avidly, forming robust biofilms on catheters implanted in mice [41].In the same context of analyzing pathogenesis, Wang TW et al. used a comprehensive transcriptional analysis to identify key cell surface adhesins.They used a mouse model of catheter infections and they identified that adhesins were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms.This could explain the flexibility of C. auris and its rapid adaptation to the environment, potentially impacting persistence and virulence [42].Magnasco L et al. was interested in the frequency of C. auris outside a highly endemic setting and, after analyzing all the cases from their 1000-bed hospital in Genoa, Italy, they recommended screening at discharge from the endemic ward(s), even in the case of a recent negative result [43].Pra ży ńska M et al. reported a case of meningitis positive for C. auris, identified by MALDI-TOF and WGS [44].Theodoropoulos NM et al. reported a cluster of six patients, two being recipients of liver transplants, positive for C. auris [45].Solid-organ transplant recipients are at high risk of developing C. auris bloodstream infections, as the authors from Florida, USA, found C. auris in five solid-organ transplant recipients (one heart, three liver, and one combined liver-kidney transplants) [46].
Regarding C. auris's involvement in dental pathology, research in this field is still in its beginning.A September 2023 study by Alfaifi et al. presented a retrospective analysis, over a period of 2 years, from the institutional experience of a single center.In this study, 310 samples of saliva from patients experiencing symptoms or who had evidence of oral candidiasis were tested for the presence of yeasts.The result was that C. auris was not isolated from any of these samples, leading to the conclusion that it is not a common colonizer of the oral cavity.Also, the authors identified eight other studies of Candida auris being detected in more than 30 ocular, nasal, pharynx, and tracheal samples [47].Another research team reviewed papers about the composition microbial biofilm found on dentures.While there is an important body of data on the microbial composition of dental plaque, the data published about denture plaque are limited.These artificial surfaces, depending on the materials in their structure, can be more prone to colonization by some microorganisms, including Candida species, leading to denture-associated stomatitis.C. albicans is the most well-studied organism found in denture plaque, followed to a lesser extent by other yeast species, and regarding C. auris, the authors note that, to date, no study has reported it to be found in denture microbial biofilms [48].Transient oral colonization with this multidrug resistant fungus, in non-human mammals, has already been reported, and the first such case of C. auris oral colonization in a pet was published in 2024.The health implication of this oral colonization in pets is that zoonotic transmission to humans may be possible, given the long period of time that this pathogen can remain on surfaces [49].In the case that human transient oral colonization is also reported, screening for oral C. auris may be recommended in the future before dental procedures, due to the possibility of hematogenous spread and even severe systemic infections in immunosuppressed patients.
This short summary showed that C. auris is capable of infecting patients from different medical wards (surgical, infectious disease, neonates, solid organ transplants, and dentistry).

Candida auris: Antifungal Resistance Mechanisms
The antifungal resistance phenomenon is well-known for Candida auris.We analyzed which were the most frequently identified resistance mechanisms in original studies published in PubMed in the last year (2023-2024).We identified 10 original papers, 4 of them published by researchers from the USA.The antifungal resistance mechanisms identified were diverse: resistance to phagocytosis by macrophages, mutations that explain increased fluconazole and voriconazole resistance (ERG11 and TAC1B genes), missense substitutions in FKS1 for echinocandin resistance, and the possibility of using the proton pump inhibitor lansoprazole to restore the activity of amphotericin B (AmB) in resistant isolates.Identifying with high precision the antifungal resistance mechanisms could lead to opportunities in the development of novel antifungal drugs against C. auris and could help in tracking the spread of Candida auris between cities and countries by highlighting the genetic relation between strains (Table 2) [14,[50][51][52][53][54][55][56][57][58].

Candida auris Antifungal Stewardship
Kriegl L et al., in a paper published in March 2024, mentioned C. auris as one of the most important WHO fungal priority pathogens.The authors presented an antifungal therapy that was shown to have a good activity against these fungi.Echinocandin therapy is currently considered the first choice when treating an invasive C. auris infection, with the novel echinocandin, rezafungin, having very good in vitro activity against C. auris.For the C. auris isolates with FKS mutations encoding for echinocandin resistance, ibrexafungerp treatment seems to be effective.Another promising therapy is the one with fosmanogepix, as it proved its efficiency in MDR (multidrug-resistant) C. auris candidemia and in mice and rabbit models of cerebral spinal fluid infection therapy [59].
Several papers reported on the relationship between the C. auris clade and antifungal resistance, which were included in a recent review by Sharma and Kadosh, who found that isolates belonging to clades I, III, IV, and V show resistance to fluconazole and crossresistance to echinocandins and amphotericin B, with even the possibility of pan-drugresistant strains.Clade II, found predominantly in Korea and Japan, was found to have a lower degree of antifungal resistance [60].
The WHO guideline mentions surveillance actions, interventions, and strategies, in which there is a crucial necessity of antifungal stewardship activities by limiting the inappropriate use of antifungals and antibiotics, avoiding antifungal's overuse and selecting the best drug, dose, and duration, and appropriate cases where they are needed.Also, it is mandatory to develop standard procedures to optimize the diagnosis of fungal infections that also include pathogens with outbreak potential and increase the capacity for outbreak detection, reporting, and response [1].

Conclusions
Candida auris was involved in more than twenty outbreaks, happening in all continents, in previous years.Given the fact that there is no specific prevention mechanism available, the fast detection of this pathogen in patient samples, without prior DNA extraction, can prove to be a salutary step in the management of these cases.In this way, by ensuring fast detection, safety procedures for limiting the spread of C. auris in the hospital environment could be implemented.From all the identified assays (e.g., WGS, MALDI-TOF, and PCR) to diagnose C. auris outbreaks, the most appropriate for routine diagnosis is, from our point of view, real-time PCR, due to its detection speed, the possibility of also identifying resistance genes, and readily available laboratory equipment.Commercially available kits should be validated by each laboratory before implementing them in routine diagnosis.Moreover, studies that already identified the resistance mechanisms to antifungals used for the treatment of these infections will favor stewardship strategies, contributing to preventing future resistance emergence.

Table 1 .
Molecular detection of Candida auris in recent worldwide outbreaks.

Table 2 .
Identified antifungal resistance mechanisms for Candida auris.