Identification of Candida auris and related species by multiplex PCR based on unique GPI protein-encoding genes.

BACKGROUND: The pathogen Candida auris is rapidly gaining clinical importance because of its resistance to antifungal treatments and its persistence in hospital environments. Early and accurate diagnosis of C. auris infections is crucial, and however, the fungus has often been misidentified by commercial systems. OBJECTIVES: To develop conventional and real-time PCR methods for accurate and rapid identification of C. auris and its discrimination from closely related species by exploiting the uniqueness of certain glycosylphosphatidylinositol (GPI)-modified protein-encoding genes. METHODS: Species-specific primers for two unique putative GPI protein-encoding genes per species were designed for C. auris, C. haemulonii, C. pseudohaemulonii, C. duobushaemulonii, C. lusitaniae and C. albicans. Primers were blind tested for their specificity and efficiency in conventional and real-time multiplex PCR set-up. RESULTS: All primers combinations showed excellent species specificity. In multiplex mode, correct identification was aided by different-sized amplicons for each species. Efficiency of the C. auris primers was validated using a panel of 155 C. auris isolates, including all known genetically diverse clades. In real-time multiplex PCR, different melting points of the amplicons allowed the distinction of C. auris from four related species. C. auris limit of detection was 5 CFU/reaction with a threshold value of 32. The method was also able to detect C. auris in spiked blood and serum. CONCLUSIONS: PCR identification based on unique GPI protein-encoding genes allows for accurate and rapid species identification of C. auris and related species without need for expensive equipment when applied in conventional PCR set-up.

Molecular identification of Candida auris by PCR amplification of species-specific GPI protein-encoding genes.

The emerging multidrug-resistant pathogenic yeast Candida auris causes life-threatening invasive infections and shows a capacity for hospital transmission that is uncommon in other Candida species. Rapid and accurate diagnosis of C. auris infections is crucial; however, the fungus is frequently misidentified. Here, we present a rapid and easily applicable PCR assay for reliable identification of C. auris by designing primers from unique GPI protein-encoding genes. Specificity of the used primers for C. auris was verified with a panel of 19 different Candida species including the clinically most relevant and phylogenetically closely related species. Efficacy of the PCR approach was validated by correctly identifying 112 C. auris isolates from an outbreak in a Spanish hospital, 20% of which were not reliably identified by MALDI-TOF MS, and 27 genotypically diverse C. auris isolates originating from hospitals in various countries, in a test that included (blind) negative controls. By employing two GPI protein primer pairs in a single PCR, a double screening can be performed, which enhances the robustness of the PCR assay and avoids potential false negatives due to recent evolutionary events, as was observed for two isolates. Our PCR method, which is based on the uniqueness of selected GPI protein-encoding genes, is useful for easy, low-cost, and accurate identification of C. auris infections in a clinical setting.

The Ecology of Non-Candida Yeasts and Dimorphic Fungi in Cetaceans: From Pathogenicity to Environmental and Global Health Implications.

Cetaceans, which are integral to marine ecosystems, face escalating anthropogenic threats, including climate change and pollution, positioning them as critical sentinel species for ocean and human health. This review explores the neglected realm of non-Candida yeasts in cetaceans, addressing the gaps in the understanding of their prevalence, pathogenicity, and environmental impacts. By examining identified species such as Cryptococcus spp., Paracoccidioides spp., and several dimorphic fungi, this review emphasizes global prevalence, epidemiology and ecology, pathogenicity, and potential zoonotic implications. It also discusses the fine line between yeast commensalism and pathogenicity by considering environmental influences such as pollution, climate shifts, and immune suppression. Environmental impact discussions delve into how rising ocean temperatures and pollution can modify yeast mycobiota, potentially affecting marine host health and broader ecosystem dynamics. The cetacean's unique physiology and ecological niches are considered, highlighting potential impacts on behaviors, reproductive success, and survival rates. Identifying crucial knowledge gaps, the review calls for intensified research efforts, employing advanced molecular techniques to unravel the cetacean mycobiome. Systematic studies on yeast diversity, antifungal susceptibility, and their influence on environmental and ecosystem health are proposed, and the balance between commensal and pathogenic species emphasizes the significance of the One Health approach. In conclusion, as marine mammals face unprecedented challenges, unveiling non-Candida yeasts in cetaceans emerges as a critical endeavor with far-reaching implications for the conservation of marine ecosystems and for both animal and human public health.

Potential Fungal Zoonotic Pathogens in Cetaceans: An Emerging Concern.

Over 60% of emerging infectious diseases in humans are zoonotic, often originating from wild animals. This long-standing ecological phenomenon has accelerated due to human-induced environmental changes. Recent data show a significant increase in fungal infections, with 6.5 million cases annually leading to 3.7 million deaths, indicating their growing impact on global health. Despite the vast diversity of fungal species, only a few are known to infect humans and marine mammals. Fungal zoonoses, especially those involving marine mammals like cetaceans, are of global public health concern. Increased human-cetacean interactions, in both professional and recreational settings, pose risks for zoonotic disease transmission. This review focuses on the epidemiology, clinical manifestations, and zoonotic potential of major fungal pathogens shared in humans and cetaceans, highlighting their interspecies transmission capability and the challenges posed by antifungal resistance and environmental changes. It underscores the need for enhanced awareness and preventative measures in high-risk settings to protect public health and marine ecosystems.

Candida spp. in Cetaceans: Neglected Emerging Challenges in Marine Ecosystems.

Cetaceans, which are crucial in marine ecosystems, act as sentinels for ecosystem and human-environmental health. However, emerging fungal infections, particularly by Candida spp., pose a growing concern in these marine mammals. This review consolidates current knowledge on the prevalence, clinical manifestations, species distribution, and antifungal resistance of Candida infections in cetaceans. We detail the diverse pathogenic impacts of Candida, including respiratory, dermal, and systemic afflictions, underscoring diagnostic and treatment challenges amid rising antifungal resistance. Our analysis extends beyond health concerns in captive cetaceans, where confinement stress heightens vulnerability, to encompass substantial ecological risks in wild populations. The review emphasizes the One Health perspective, linking cetacean health with broader environmental and human public health issues. We particularly focus on the potential zoonotic transmission of emerging fungal pathogens such as Candida auris and the role of environmental changes in fostering antifungal resistance. The study underscores the need for concerted, interdisciplinary efforts in veterinary, medical, and environmental sciences to enhance understanding and management of Candida infections in cetaceans. We advocate for comprehensive monitoring and collaborative research initiatives to mitigate the rising challenge of these infections. Addressing Candida spp. in cetaceans is not just a conservation priority but a critical step in safeguarding overall marine health and, by extension, human health in the context of evolving infectious diseases.

Outbreak of Candida auris in Spain: A comparison of antifungal activity by three methods with published data.

Candida auris is an emerging pathogen causing candidaemia outbreaks in several countries for which azole, amphotericin B (AmB) and echinocandin resistance has been reported. In this study, the antifungal susceptibilities of 73 Spanish C. auris isolates (56 bloodstream and 17 urine) to eight antifungal agents were determined using three methods. Isolates were identified by internal transcribed spacer (ITS) sequencing, and minimum inhibitory concentrations (MICs) of fluconazole, isavuconazole, itraconazole, posaconazole, voriconazole, anidulafungin, micafungin and AmB were determined by EUCAST methodology and Sensititre® YeastOne® (SYO) (bloodstream isolates) and Liofilchem® MIC Test Strip (all isolates). Agreement between the methods was analysed and the MICs (ours and published data) were categorised using recently proposed epidemiological cut-off values (ECVs). Fluconazole MICs were >64 mg/L, whilst >60% of voriconazole MICs were >1 mg/L by the three methods. Posaconazole was the most active azole (EUCAST geometric mean MIC, 0.053 mg/L), followed by isavuconazole (0.066 mg/L) and itraconazole (0.157 mg/L). Echinocandins MICs were ≤0.5 mg/L by SYO and EUCAST. The overall lowest AmB MICs (≤0.25 mg/L) were obtained by EUCAST. Essential agreement (±2 dilutions) between EUCAST and SYO was >93% for the eight antifungals. For this new C. auris clade, all isolates were resistant to fluconazole, and MICs for anidulafungin, micafungin and AmB were ≤1 mg/L using dilution methods. Voriconazole MICs were method-dependent. The number of non-wild-type (non-WT) isolates depended on the ECV applied; by the 97.5% ECV all isolates were WT except for isavuconazole (1.8% non-WT). Good essential agreement (>93%) was observed between EUCAST and SYO.

Nosocomial fungemia by Candida auris: First four reported cases in continental Europe.

BACKGROUND: Candida auris is an emerging multidrug-resistant yeast that can cause invasive infections and is associated with high mortality. It is typically resistant to fluconazole and voriconazole and, some cases, also to echinocandins and amphotericin B. This species, phylogenetically related to Candida haemulonii, is frequently misidentified by commercial identification techniques in clinical laboratories; therefore, the real prevalence of C. auris infections may be underestimated. AIMS: To describe the clinical and microbiological features of the first four cases of C. auris fungemia episodes observed in the European continent. METHODS: The four patients were hospitalized in the adult surgical intensive care unit. A total of 8 isolates (two per patient) from blood and catheter tip were analyzed. RESULTS: All isolates were misidentified as Saccharomyces cerevisiae by AuxaColor 2, and as Candida sake by API ID20C. VITEK MS technology misidentified one isolate as Candida lusitaniae, another as C. haemulonii and could not identify the other six. C. auris identification was confirmed by ITS rDNA sequencing. All isolates were fluconazole (MIC >256mg/l) and voriconazole (MIC 2mg/l) resistant and susceptible to posaconazole, itraconazole, echinocandins and amphotericin B. CONCLUSIONS: C. auris should be regarded as an emerging pathogen, which requires molecular methods for definitive identification. Our isolates were highly resistant to fluconazole and resistant to voriconazole, but susceptible to the other antifungals tested, which emphasizes the importance of accurately identifying this species to avoid therapeutic failures.