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Eukaryotic genomes typically encode one member of the DXO/Dxo1/Rai1 family of enzymes, which can hydrolyze the 5' ends of RNAs with a variety of structures that deviate from the canonical 7m GpppN. In contrast, the Saccharomyces genome encodes two family members and the second copy, Dxo1, is a distributive 5' exoribonuclease that is required for the final maturation of the 5' end of 25S rRNA from a 25S' precursor. Here we show that this 25S rRNA maturation function is not conserved across kingdoms, but arose in the budding yeasts. Interestingly, the origin of 25S processing capacity coincides with the duplication of this gene, and this capacity is absent in the nonduplicated genes. Strikingly, two different clades of budding yeasts have undergone parallel evolution: Both duplicated their DXO/Dxo1/Rai1 gene, and in both cases, one copy gained the 25S processing function. This was accompanied by many parallel sequence changes, a remarkable case of reproducible neofunctionalization., (© 2024 Hurtig et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Amphotericin B (AmpB) is an effective but toxic antifungal drug. Thus, improving its activity/toxicity relationship is of interest. AmpB disrupts fungal membranes by two proposed mechanisms: ergosterol sequestration from the membrane and pore formation. Whether these two mechanisms operate in conjunction and how they could be potentiated remains to be fully understood. Here, we report that gladiolin, a polyketide antibiotic produced by Burkholderia gladioli , is a strong potentiator of AmpB and acts synergistically against Cryptococcus and Candida species, including drug-resistant C. auris . Gladiolin also synergizes with AmpB against drug-resistant fungal biofilms, while exerting no mammalian cytotoxicity. To explain the mechanism of synergy, we show that gladiolin interacts with membranes via a previously unreported binding mode for polyketides. Moreover, gladiolin modulates lipid binding by AmpB and, in combination, causes faster and more pronounced lipid rearrangements relative to AmpB alone which include membrane thinning consistent with ergosterol extraction, areas of thickening, pore formation, and increased membrane destruction. These biophysical data provide evidence of a functional interaction between gladiolin and AmpB at the membrane interface. The data further indicate that the two proposed AmpB mechanisms (ergosterol sequestration and pore formation) act in conjunction to disrupt membranes, and that gladiolin synergizes by enhancing both mechanisms. Collectively, our findings shed light on AmpB's mechanism of action and characterize gladiolin as an AmpB potentiator, showing an antifungal mechanism distinct from its proposed antibiotic activity. We shed light on the synergistic mechanism at the membrane, and provide insights into potentiation strategies to improve AmpB's activity/toxicity relationship., Importance: Amphotericin B (AmpB) is one of the oldest antifungal drugs in clinical use. It is an effective therapeutic, but it comes with toxicity issues due to the similarities between its fungal target (the membrane lipid ergosterol) and its mammalian counterpart (cholesterol). One strategy to improve its activity/toxicity relationship is by combinatorial therapy with potentiators, which would enable a lower therapeutic dose of AmpB. Here, we report on the discovery of the antibiotic gladiolin as a potentiator of AmpB against several priority human fungal pathogens and fungal biofilms, with no increased toxicity against mammalian cells. We show that gladiolin potentiates AmpB by increasing and accelerating membrane damage. Our findings also provide insights into the on-going debate about the mechanism of action of AmpB by indicating that both proposed mechanisms, extraction of ergosterol from membranes and pore formation, are potentiated by gladiolin., Competing Interests: G.L.C. is a shareholder, non-executive director, and consultant of Erebagen Ltd. The other authors declare no competing interests.

Vaginal candidiasis (VC) is a prevalent condition among women of reproductive age and poses a significant global public health challenge. However, the disease is often diagnosed and treated without mycological information. We investigated the epidemiology, laboratory diagnostics, and antifungal susceptibility of VC. We included 300 women from Çukurova University Obstetrics and Gynecology outpatient clinic in Adana, Türkiye. Participants underwent a health survey and provided vaginal swab samples for microscopic examination and fungal culture. The microscopic analysis involved wet-mount and gram-stained slides, whereas fungal identification involved CHROMAgar Candida, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and real-time polymerase chain reaction high-resolution melting analysis (RT-PCR HRMA). Antifungal susceptibility tests were conducted at pH 7 and 4 using the CLSI document M44-A2. Of the 106 women with positive fungal cultures, 86.8% were diagnosed with VC, whereas 13.2% showed Candida colonization. Among those with VC, 55.4% had acute and 44.6% had recurrent VC; a family history of allergies increased the risk for both types. We recovered 115 yeast isolates, predominantly C. albicans, C. glabrata, and C. krusei. Diagnostic accuracy of CHROMAgar Candida was 91.3% for the most common isolates, and HRMA was consistent in differential diagnosis. Antifungal resistance varied with pH; susceptibility to fluconazole, itraconazole, and ketoconazole decreased at pH 4, whereas susceptibility to miconazole increased. Our findings underscore the need for a diagnostic algorithm and enhanced collaboration between clinicians and microbiologists to improve VC management. Recommendations include using Gram staining, CHROMAgar Candida, MALDI-TOF MS, and antifungal susceptibility tests at both pH levels., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: The Nakaseomyces clade is formed by at least nine described species among which three can be pathogenic to humans, namely Nakaseomyces glabratus (Candida glabrata), the second most-common cause of candidiasis worldwide, and two rarer emerging pathogens: Nakaseomyces (Candida) nivarensis and Nakaseomyces (Candida) bracarensis. Early comparative genomics analyses identified parallel expansions of subtelomeric adhesin genes in N. glabratus and N. nivarensis/bracarensis, and suggested possible links with the emergence of the virulence potential in these species. However, as shown for N. glabratus, the proper assessment of subtelomeric genes is hindered by the use of incomplete assemblies and reliance on a single isolate., Results: Here we sequenced seven N. bracarensis isolates and reconstructed chromosome level assemblies of two divergent strains. We show that N. bracarensis isolates belong to two diverging clades that have slightly different genomic structures. We identified the set of encoded adhesins in the two complete assemblies, and uncovered the presence of a novel adhesin motif, found mainly in N. bracarensis. Our analysis revealed a larger adhesin content in N. bracarensis than previously reported, and similar in size to that of N. glabratus. We confirm the independent adhesin expansion in these two species, which could relate to their different levels of virulence., Conclusion: N. bracarensis clinical isolates belong to at least two differentiated clades. We describe a novel repeat motif found in N. bracarensis adhesins, which helps in their identification. Adhesins underwent independent expansions in N. glabratus and N. bracarensis, leading to repertoires that are qualitatively different but quantitatively similar. Given that adhesins are considered virulence factors, some of the observed differences could contribute to variations in virulence capabilities between N. glabratus and N. bracarensis., (© 2024. The Author(s).)

Vaginitis is a prevalent clinical disorder associated with several adverse health consequences, prompting women to seek medical care. In this study we evaluate the Vaginal Panel Real-Time PCR kit (qPCR test) against routinely used diagnostics for detection of bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), and trichomoniasis. A total of 1011 vaginal swab specimens were analyzed. The routinely diagnostic methods for BV was Gram stain-based Nugent score. VVC presence was detected by culture, and Candida species were identified using MALDI-TOF MS. Trichomonas vaginalis was identified by culture in a selective medium. Molecular analyses were conducted on the MagXtract® 3200 System and analyzed using the CFX96™ Real-Time PCR Detection System. The sensitivity, specificity, positive predictive value, and negative predictive value of the qPCR test compared to the reference method for BV diagnosis was 93.1%, 88.8%, 90.1% and 92.2%, respectively, with a Kappa value of 0.82. For Candida species, sensitivity, specificity, positive predictive value, and negative predictive value were 96.0%, 98.4%, 95.3%, and 98.7%, respectively. The qPCR test detected 32 additional positive samples for Candida not reported by the routinely used diagnostics. For trichomoniasis, the qPCR test identified T. vaginalis in fifteen specimens, despite no microscopic detection in cultured specimens. Our results demonstrate that the Vaginal Panel Real-Time PCR kit shows optimal concordance with routinely used diagnostics for diagnosing vaginitis. Furthermore, enhancing detection of T. vaginalis. However, further validation studies are necessary to confirm its full diagnostic accuracy. The use of nucleic acid amplification tests (NAATs) provides rapid and accurate diagnosis, crucial for early detection and treatment of vaginitis., Competing Interests: The funder provided support in the form of salaries for authors IA, SUP and PM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Amor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Abdominal aortic aneurysm (AAA) is a large-vessel disease with high mortality, characterized by complex pathogenic mechanisms. Current therapeutic approaches remain insufficient to halt its progression. Fungi are important members of the gut microbiota. However, their characteristic alterations and roles in AAA remain unclear. This study investigated the role of gut fungal communities in the development of AAA through metagenomic sequencing of fecal samples from 31 healthy individuals and 33 AAA patients. We observed significant dysbiosis in the gut mycobiomes of AAA patients compared to healthy individuals, characterized by an increase in pathogenic fungi like Candida species and a decrease in beneficial yeasts such as Saccharomyces cerevisiae . The changes in fungal populations correlated strongly with clinical indicators of AAA, highlighting their potential for diagnosing and predicting AAA progression. Furthermore, our animal experiments demonstrated that Saccharomyces cerevisiae significantly ameliorated pathological alterations in AAA mice, suggesting a protective role for specific yeast strains against AAA development. These findings underscore the significant impact of gut mycobiomes on AAA and suggest that modulating these fungal communities could offer a novel therapeutic approach. Our research advances the understanding of the influence of gut microbiome on vascular diseases and suggests potential non-surgical approaches for managing AAA. By elucidating the diagnostic and therapeutic potential of gut fungi in AAA, this study provided important clues for future clinical strategies and therapeutic developments in the field of vascular medicine., Importance: Our research highlights the crucial role of gut fungi in abdominal aortic aneurysm (AAA) development. By analyzing fecal samples from AAA patients and healthy controls, we discovered significant dysbiosis in gut fungal communities, characterized by an increase in harmful Candida species and a decrease in beneficial yeasts like Saccharomyces cerevisiae . This dysbiosis was correlated with the severity of AAA. Importantly, in animal experiments, supplementing with Saccharomyces cerevisiae significantly slowed AAA progression. These findings suggest that modulating gut fungi may offer a novel, non-surgical approach to the diagnosis and treatment of AAA, potentially reducing the need for invasive procedures., Competing Interests: The authors declare no conflict of interest.

Objectives: Ascites, often associated with critical pathologies such as liver cirrhosis or bowel perforation, can be complicated by fungal infection, increasing mortality especially in intensive care settings and demanding rapid diagnosis and adequate treatment. Traditional microbiological diagnostic methods have limited sensitivity in accurately identifying fungal pathogens in ascitic fluid. Alternative diagnostic methods may offer important insights to enable guiding of antifungal therapy and refining empirical treatment strategies. The objective of this study was to evaluate the potential of next-generation sequencing methods to identify specific fungal pathogens responsible for ascitic fluid infections., Methods: We prospectively collected 50 ascitic fluid samples from ICU patients with suspected ascites infection. In addition to standard culture-based microbiological testing, an ascitic fluid aliquot underwent fungal DNA isolation and was analyzed by next-generation sequencing (NGS) methods for identification of fungal species., Results: Of 50 ascitic samples collected, five samples showed growth of Candida spp. in culture. After DNA isolation and ITS2 PCR, detectable amplification was achieved in 10 samples. Sequencing of the 50 patients' samples identified facultative pathogenic fungi in 19 patients. In 15 cases, culture alone would not have permitted the identification of all facultative pathogenic fungi. The identification of fungal DNA by sequencing was significantly associated with poor patient outcome and a number of clinical parameters., Conclusions: Our results show a higher sensitivity for NGS-based diagnostic methods in the identification of ascitic fluid fungal infections compared to culture-based diagnostics. This may be beneficial especially for patients in a critical care setting, who have an increased prevalence of comorbidities and high mortality. The implementation of such methods in standard diagnosis will require increased standardization of the workflows and interpretation of the sequencing results with respect to patients' clinical picture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Posadas-Cantera, Mehrbarzin, Wetzel, Goelz, Kousoulas, Utzolino, Häcker and Badr.)

Introduction: Candida auris is an emerging pathogen that represents a worldwide health problem due to its global expansion, multidrug resistance, and difficult laboratory identification. Among the risk factors for colonization/infection by C. auris , a stay in an intensive care unit (ICU) stands out. This prospective multicenter study aimed to monitor the trend of the local epidemiology of Candida spp. and unveil the prevalence of C. auris ., Methods: From 2020 to 2022, axillar/inguinal swabs were collected from adult patients at three points: upon admission (D1) and on the fifth (D5) and eighth (D8) days of their ICU stay. We employed culture-based screening methods combined with molecular techniques to identify Candida spp. down to the species level. Specific screening for Candida auris was conducted using a real-time PCR assay in combination with an improved selective culture medium, mannitol salt agar auris (MSAA). To validate the effectiveness of MSAA, a collection of reference C. auris strains representing the four major geographical clades was used., Results: We enrolled 675 patients, and 355 Candida isolates were retrieved from the 988 swab samples collected. From those, 185/355 (52.1%) were identified as C. albicans and 170/355 (47.9%) as non- albicans Candida (NAC). MSAA medium showed a specificity of 94.8%, albeit C. auris was not detected in this cohort. The dynamics of Candida spp. colonization by ICU were significant at the three collection points. Upon admission, C. albicans was associated with the Beatriz Ângelo Hospital ICU ( p =0.003) and C. tropicalis with the general Hospital Professor Doutor Fernando Fonseca (FFH) ICU ( p =0.006). C. parapsilosis and C. lusitaniae were associated with FFH ICUs, with the general ICU at D5 ( p =0.047) and surgical ICU at D8 ( p =0.012). The dynamics of NAC colonization by ICU were significantly different at D1 ( p =0.011), D5 ( p =0.047), and D8 ( p =0.012)., Conclusion: We developed and implemented a screening protocol for C. auris while uncovering the colonization patterns of Candida in the ICU. Our findings contribute to the optimization of overall patient management, ensuring that ICU protocols are resilient and adaptive to emerging fungal threats., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nascimento, Inácio, Guerreiro, Diaz, Patrício, Proença, Toscano and Barroso.)

Antifungal drug resistance represents a serious global health threat, necessitating new treatment strategies. Here we investigated collateral sensitivity (CS), in which resistance to one drug increases sensitivity to another, and cross-resistance (XR), in which one drug resistance mechanism reduces susceptibility to multiple drugs, since CS and XR dynamics can guide treatment design to impede resistance development, but have not been systematically explored in pathogenic fungi. We used experimental evolution and mathematical modelling of Candida auris population dynamics during cyclic and combined drug exposures and found that especially CS-based drug cycling can effectively prevent the emergence of drug resistance. In addition, we found that a CS-based treatment switch can actively select against or eradicate resistant sub-populations, highlighting the potential to consider CS in therapeutic decision-making upon resistance detection. Furthermore, we show that some CS trends are robust among different strains and resistance mechanisms. Overall, these findings provide a promising direction for improved antifungal treatment approaches., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Lactose assimilation is a relatively rare trait in yeasts, and Kluyveromyces yeast species have long served as model organisms for studying lactose metabolism. Meanwhile, the metabolic strategies of most other lactose-assimilating yeasts remain unknown. In this work, we have elucidated the genetic determinants of the superior lactose-growing yeast Candida intermedia . Through genomic and transcriptomic analyses, we identified three interdependent gene clusters responsible for the metabolism of lactose and its hydrolysis product galactose: the conserved LAC cluster ( LAC12 , LAC4 ) for lactose uptake and hydrolysis, the conserved GAL cluster ( GAL1 , GAL7 , and GAL10 ) for galactose catabolism through the Leloir pathway, and a " GALLAC " cluster containing the transcriptional activator gene LAC9 , second copies of GAL1 and GAL10 , and a XYL1 gene encoding an aldose reductase involved in carbon overflow metabolism. Bioinformatic analysis suggests that the GALLAC cluster is unique to C. intermedia and has evolved through gene duplication and divergence, and deletion mutant phenotyping proved that the cluster is indispensable for C. intermedia's growth on lactose and galactose. We also show that the regulatory network in C. intermedia , governed by Lac9 and Gal1 from the GALLAC cluster, differs significantly from the galactose and lactose regulons in Saccharomyces cerevisiae , Kluyveromyces lactis , and Candida albicans . Moreover, although lactose and galactose metabolism are closely linked in C. intermedia , our results also point to important regulatory differences.IMPORTANCEThis study paves the way to a better understanding of lactose and galactose metabolism in the non-conventional yeast C. intermedia . Notably, the unique GALLAC cluster represents a new, interesting example of metabolic network rewiring and likely helps to explain how C. intermedia has evolved into an efficient lactose-assimilating yeast. With the Leloir pathway of budding yeasts acting like a model system for understanding the function, evolution, and regulation of eukaryotic metabolism, this work provides new evolutionary insights into yeast metabolic pathways and regulatory networks. In extension, the results will facilitate future development and use of C. intermedia as a cell-factory for conversion of lactose-rich whey into value-added products., Competing Interests: The authors declare no conflict of interest.

We measured concentrations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, influenza A and B viruses, respiratory syncytial virus, human metapneumovirus, enterovirus D68, human parainfluenza types 1, 2, 3, 4a, and 4b in aggregate, norovirus genotype II, rotavirus, Candida auris, hepatitis A virus, human adenovirus, mpox virus, H5 influenza A virus, and pepper mild mottle virus nucleic acids in wastewater solids prospectively at 191 wastewater treatment plants in 40 states across the United States plus Washington DC. Measurements were made two to seven times per week from 1 January 2022 to 30 June 2024, depending on wastewater treatment plant staff availability. Measurements were made using droplet digital (reverse-transcription-) polymerase chain reaction (ddRT-PCR) following best practices for making environmental molecular biology measurements. These data can be used to better understand disease occurrence in communities contributing to the wastewater., (© 2024. The Author(s).)

The use of recreational waters is a widespread activity worldwide, and one of the risks associated with this practice is the exposure of bathers to microorganisms that may arise due to pollution caused by inadequate infrastructure and sanitation. In the present work, we isolated Candida spp. (n = 24) from five recreational beaches in Rio de Janeiro, Brazil, in order to evaluate their susceptibility to antifungals, the production of virulence attributes and the in vivo virulence using Tenebrio molitor larvae as a model. The ITS1-5.8S-ITS2 gene sequencing identified thirteen isolates (54.1 %) as C. tropicalis, seven (29.1 %) as C. krusei (Pichia kudriavzevii), one (4.2 %) as C. rugosa (Diutina rugosa), one (4.2 %) as C. mesorugosa (Diutina mesorugosa), one (4.2 %) as C. utilis (Cyberlindnera jadinii) and one (4.2 %) as C. parapsilosis. C. tropicalis isolates showed resistance to azoles and susceptibility to amphotericin B, flucytosine and caspofungin. C. krusei isolates were resistant to fluconazole, caspofungin and itraconazole, with 42.8 % resistance to flucytosine, besides susceptibility to voriconazole and amphotericin B. The remaining species were susceptible to all tested antifungals. All Candida isolates adhered to abiotic surfaces and formed biofilm on polystyrene, albeit to varying degrees, and produced aspartic protease and hemolytic activity, which are considered fungal virulence attributes. C. tropicalis, C. krusei and C. utilis isolates produced phytase, while the only esterase producer was C. tropicalis. Regarding resistance to osmotic stress, all isolates of C. tropicalis, C. parapsilosis and C. mesorugosa grew up to 7.5 % NaCl; the remaining isolates grew up to 1.87-3.75 % NaCl. The mortality caused by fungal challenges in T. molitor larvae was variable, with C. tropicalis, C. utilis and C. parapsilosis being more virulent than C. krusei and C. rugosa complex. Collectively, the presence of these yeasts, particularly the virulent and resistant isolates, in recreational waters can pose a significant health risk to bathers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Candida spp. are the most common cause of opportunistic fungal infections and are associated with a high mortality rate worldwide. In Palestine, the prevalence of Candida spp. infections remains elusive., Methods: We performed our study at two hospitals in Palestine (Istishari Arab Hospital, and Najah National University Hospital). All patients diagnosed with candidiasis during the year 2022 have participated in the study. The prevalence of Candida spp., their distribution, and the activity of selected antifungals against Candida pathogens were assessed. In combination with phenotypic properties, Candida isolates were identified and tested for antifungal susceptibility using the colorimetric VITEK-2 Compact system., Results: Our results showed that the prevalence of Candida spp. among infected samples was 11.6%. A total of eleven different Candida spp. were identified. Among these isolates, C. albicans (46.54%) was the most frequent, followed by C. glabrata (16.14%), C. tropicalis (13.83%), C. parapsilosis (4.82%), C. krusei (3.56%), C. dubliniensis (2.09%), C. ciferrii (1.67%), C. lusitaniae (0.83%), C. guilliermondii (0.62%), C. kefyer (0.41%) and C. spherica (0.20%). Among C. albicans, all isolates were 100% susceptible to fluconazole and micafungin. The susceptibility rates to Amphotericin B and flucytosine were 95% and 99%, respectively. The susceptibility rates of non-albicans Candida spp. (NAC) to fluconazole, voriconazole, amphotericine B, caspofungin, flucytosine and micafungin were 70%, 99%, 97%, ,72%, 92% and 100%, respectively. The incidence of Candida infections was higher in the intensive care unit and surgery department as compared to other hospital departments., Conclusions: Four pathogens are responsible for the most invasive infections: C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis. A notable characteristic of this study was the high frequency of NAC species which were often more resistant to antifungal agents. A quick and accurate system like Vitek 2 compact was suggested for the careful species identification of clinical isolates of Candida. We suggest that continued surveillance of species distribution and susceptibility to antifungals will enhance future burden estimates and assist in evaluating preventative measures' effectiveness., (© 2024. The Author(s).)

Background: We report two cases of fungal endophthalmitis induced by Candida species identified based on internal transcribed spacer 1 (ITS1) sequencing., Case Presentation: In two cases, endophthalmitis was suspected, and the patients underwent pars plana vitrectomy. Case 1 was a 64-year-old woman with a history of cataract surgery 10 days prior. She had a history of anal primary melanoma, which metastasized throughout the body and subsequently relapsed. Vitreous culture and ITS-1 deep sequencing revealed the presence of the rare fungus, Candida dubliniensis. Case 2 was a 54-year-old man with a history of liver cancer and kidney failure. Culture methods and ITS1 deep sequencing both revealed the presence of Candida albicans. Both patients exhibited good visual prognoses after treatment with topical and systemic antibiotics., Conclusions: We present two cases of fungal endophthalmitis caused by two Candida species identified by both the culture method and ITS1 deep sequencing. The fungal pathogen was identified by ITS deep sequencing three days after sample submission; the culture method yielded results after 1 week. These findings support the applicability of ITS1 sequencing for timely pathogen identification for cases of fungal endophthalmitis and provide detailed taxonomic information at the species level., (© 2024. The Author(s).)

We observed an increase in the frequency of Candida auris among invasive candidiasis isolates in the 2022 SENTRY Antifungal Surveillance Program compared to prior years: ≤0.1% before 2018, 0.4%-0.6% from 2018 to 2021, and 1.6% in 2022. C. auris isolates were collected in seven countries, but 28 (35.9%) isolates were recovered in the USA (five states; more common in New York, Texas, and New Jersey) and 26 (33.3%) in Panama. Greece and Turkey had 12 and 9 isolates, respectively. Overall, 82.1% of the isolates were resistant to fluconazole; 17.9% were resistant to amphotericin B; and 1.3% were resistant to caspofungin, anidulafungin, or micafungin (Centers for Disease Control and Prevention tentative resistance breakpoints). Rezafungin inhibited 96.2% of the isolates (Clinical and Laboratory Standards Institute susceptibility breakpoint). Pandrug resistance was not observed, but 17.9% of the isolates were resistant to fluconazole and amphotericin B. South Asian (Clade I) isolates were most common ( n = 40, 51.3%); of these, 97.5% were resistant to fluconazole and 30.0% were resistant to amphotericin B. Thirty (38.5%) isolates belonged to the South American region (Clade IV), and 56.7% of those were resistant to fluconazole and 6.7% to amphotericin B. Seven isolates belonged to the South African Clade III and one to East Asian Clade II. Erg11 (Y132F, K143R, and F126L) and MRR1 (N647T) alterations were detected. One isolate that was resistant to all echinocandins carried an FKS R1354G alteration. Two isolates displayed elevated rezafungin minimum inhibitory concentration (MIC) values but low MIC values against other echinocandins and no FKS alterations. As C. auris is spreading globally, monitoring this species is prudent., Competing Interests: M.C., L.M.D., P.R.R., and C.G.C. were employees at Element Iowa City (JMI Laboratories), where the study was performed. This study was supported by Melinta Therapeutics, which included funding for services related to preparing the manuscript.

Background: Candida species have emerged as a significant cause of opportunistic infections. Alongside the expression of various virulence factors, the rise of antifungal resistance among Candida species presents a considerable clinical challenge., Aim: This study aimed to identify different Candida species isolated from clinical specimens, evaluate their antifungal sensitivity patterns, identify key genes regulating virulence mechanisms using multiplex PCR and to assess any correlation between their virulence profiles and antifungal resistance patterns., Method: A total of 100 Candida spp. was isolated from 630 different clinical specimens and identified to the species level. Their antifungal susceptibility was phenotypically evaluated in accordance with CLSI guidelines using the Vitek-2 Compact System. Virulence markers, including biofilm formation capacity, protease production, melanin production, coagulase production and hemolysin production, were also phenotypically detected. The genetic determinants for biofilm formation and extracellular hydrolytic enzymes were assessed using a multiplex PCR assay., Results: The prevalence of Candida spp. was 15.9%, with C. albicans (48%) and C. glabrata (16%) being the most common. C. albicans showed the highest virulence, with strong biofilm formation, and high proteinase and melanin production. Multiplex PCR revealed Hlp in 22.0%, Hwp in 80.0%, Als in 56.0%, and Sap genes in 56.0% of isolates. Virulence genes were more common in C. albicans than in non-albicans Candida (NAC). Resistance patterns significantly correlated with virulence profiles, with notable associations between flucytosine resistance and the presence of Hlp and Hwp genes., Conclusion: The significant correlation between virulent markers such as germination, coagulase, hemolysin production and resistance patterns among different Candida isolates is crucial for predicting the severity and outcomes of Candida infections. This understanding aids in guiding tailored treatment strategies., (© 2024. The Author(s).)

Four yeast isolates obtained from tree bark and fermenting sap of Quercus spp. and insects in Colombia and Japan were phylogenetically related to Candida galis based on analyses of the sequences of the internal transcribed spacer (ITS) region and the D1/D2 domains of the large subunit rRNA gene. The novel species differs from C. galis by 20 nt substitutions and 5 indels in the D1/D2 sequences. A phylogenomic analysis suggested that these species are related to Candida ficus , the genus Phaffomyces and a small clade containing Barnettozyma botsteinii , Barnettozyma siamensis and Candida montana . Our genomic analyses suggest that the novel species and C. galis should be separated in a novel yeast genus. We propose the genus Millerago gen. nov. to accommodate these species and the species Millerago phaffii f.a., sp. nov. (CBS 18021 T ; MycoBank MB856172) to accommodate the Colombian and Japanese isolates. The Colombian isolate of M. phaffii differs from the Japanese isolates by three nt substitutions and one indel and two substitutions and one indel in the ITS and D1/D2 sequences, respectively, showing that they were conspecific. We also propose the new species Millerago galiae sp. nov. to validate this species according to the rules of the International Code of Nomenclature for algae, fungi and plants.

Introduction . Fungal infections are relevant health risks for individuals with acquired immunodeficiency in the resource-limited tropics, but available surveillance data are scarce. For Candida auris and Cryptococcus spp., the evolution from environmental reservoirs to human pathogens causing life-threatening diseases is currently discussed as a public health concern in the context of climate change and limited treatment options. Gap statement . Uncovering the gastrointestinal tract as an epidemiological niche of fungi emerging from the environment into individuals for whom fungal infections are not diagnosed. Aim . To contribute to data on the local epidemiology of C. auris and Cryptococcus spp. in Western African Ghana by analysing gastrointestinal samples of Ghanaian individuals. Methodology . Four real-time PCR assays targeting C. auris and five real-time PCR assays targeting Cryptococcus spp. were applied with stool samples of 875 non-age-stratified Ghanaian HIV patients and 30 Ghanaian control individuals without known HIV infection. Also, 664 samples from Ghanaian children under 2 years of age were investigated. The true abundance of the target micro-organism was considered as unlikely in the case of one or fewer positive signals, likely in the case of two to three positive signals and highly likely in the case of four or more positive signals per sample in the real-time PCR assays. Results . The combined application of sensitive, target-specific real-time PCR assays indicates that neither C. auris , Cryptococcus neoformans complex nor Cryptococcus gattii complex were part of the gut microbiota of Ghanaian individuals with or without HIV infection. Conclusion . Despite the significant disease burden from these pathogens in immunosuppressed Ghanaian individuals, detection from gastrointestinal samples was unlikely, which should be taken into account when discussing screening strategies for these fungi of public health concern. In contrast, the detection of these fungi from such samples should not routinely be considered as commensal colonization flora.

The hydrolysis of cellulose generates inhibitors like acetate, suppressing fermentation performance. Here, 25SrRNA methyltransferase CgBMT5 from stress-tolerant yeast Candida glycerinogenes was used as an anti-stress gene element in Saccharomyces cerevisiae and Yarrowia lipolytica. Expression of CgBMT5 in S. cerevisiae increased cell tolerance to acetate, high osmolarity, and heat stress and rescued the delay in cell growth under acetate stress. Ethanol productivity was improved from 0.52 g·(L/h) to 0.69 g·(L/h). CgBMT5 improved GFP expression. The transcription factor ARG81 binds to the promoter of CgBMT5. CgBMT5 upregulated HOG1, GPD1, HAA1, and PMA1 and reduced ROS level, thereby improving cell resistance to acetate. CgBMT5 also improved resistance of Y. lipolytica Po1g to multiple-stress. The lipid titer was improved by 37% in the typical medium. Y. lipolytica-CgBMT5 produced 94 mg/L lipid in the undetoxified cellulose hydrolysate., (© 2024 Wiley‐VCH GmbH.)

Background: The incidence of fungal urinary tract infections (UTIs) has dramatically increased in the past decades, with Candida arising as the predominant etiological agent. Managing these infections poses a serious challenge to clinicians, especially with the emergence of fluconazole-resistant (FLC-R) Candida species. In this study, we aimed to determine the mechanisms of fluconazole resistance in urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt, assess the correlation between fluconazole resistance and virulence, and explore potential treatment options for UTIs caused by FLC-R Candida strains., Results: Fluconazole susceptibility testing of 34 urinary Candida isolates indicated that 76.5% were FLC-R, with a higher prevalence of resistance recorded in non-albicans Candida spp. (88.9%) than in Candida albicans (62.5%). The calculated Spearman's correlation coefficients implied significant positive correlations between fluconazole minimum inhibitory concentrations and both biofilm formation and phospholipase production. Real-time PCR results revealed that most FLC-R isolates (60%) significantly overexpressed at least one efflux pump gene, while 42.3% significantly upregulated the ERG11 gene. The most prevalent mutation detected upon ERG11 sequencing was G464S, which is conclusively linked to fluconazole resistance. The five repurposed agents: amikacin, colistin, dexamethasone, ketorolac, and sulfamethoxazole demonstrated variable fluconazole-sensitizing activities in vitro, with amikacin, dexamethasone, and colistin being the most effective. However, the fluconazole/colistin combination produced a notable reduction (49.1%) in bladder bioburden, a 50% decrease in the inflammatory response, and tripled the median survival span relative to the untreated murine models., Conclusions: The fluconazole/colistin combination offers a promising treatment option for UTIs caused by FLC-R Candida, providing an alternative to the high-cost, tedious process of novel antifungal drug discovery in the battle against antifungal resistance., (© 2024. The Author(s).)

Candida auris is considered a nosocomial pathogen of high concern and is currently spreading across the United States. Infection control measures for C. auris focus mainly on healthcare facilities, yet transmission levels may already be significant in the community before outbreaks are detected in healthcare settings. Wastewater-based epidemiology (culture, quantitative PCR, and whole-genome sequencing) can potentially gauge pathogen transmission in the general population and lead to early detection of C. auris before it is detected in clinical cases. To learn more about the sensitivity and limitations of wastewater-based surveillance, we used wastewater-based methods to detect C. auris in a southern Utah jurisdiction with no known clinical cases before and after the documented transfer of colonized patients from bordering Nevada. Our study illustrates the potential of wastewater-based surveillance for being sufficiently sensitive to detect C. auris transmission during the early stages of introduction into a community.

Candida infections are growing all over the world as a result of their resistance to anti-fungal drugs. This raises concerns about public health, particularly in cases of vulvovaginal candidiasis (VVC). Therefore, the need for effective treatment options for Candida infections has become crucial. The main goal of the study is to evaluate the efficacy of novel palladium metal complexes against fluconazole-resistant Candida spp., particularly C. albicans and C. auris. The process begins with identifying the minimum inhibitory concentration (MIC), followed by growth curve assays, colony morphology analysis, characterization, and gene expression analysis. The investigation revealed that sub-MIC of Pd(II) complex B (250 μg/mL) inhibited Candida spp. more effectively than amphotericin B (500 μg/mL). Further, Pd(II) complex B drastically reduced the growth of Candida spp. biofilms by 70-80% for nascent biofilms and 70-75% for mature biofilms. Additionally, the yeast-to-hyphal switch and SEM studies revealed that Pd(II) complex B effectively hinders the growth of drug-resistant Candida cells. The gene expression investigation also evidenced that Pd(II) complex B downregulated virulence genes in C. albicans (ERG, EFG, UME6, and HGC) and C. auris (ERG, CDR, and HGC). The findings showed that Pd(II) complex B effectively inhibited the growth of Candida biofilm formation and was reported as a potential anti-biofilm agent against Candida spp. that are resistant to drugs., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

A single strain of Candida anglica, isolated from cider, is available in international yeast collections. We present here seven new strains isolated from French PDO cheeses. For one of the cheese strains, we achieved a high-quality genome assembly of 13.7 Mb with eight near-complete telomere-to-telomere chromosomes. The genomes of two additional cheese strains and of the cider strain were also assembled and annotated, resulting in a core genome of 5966 coding sequences. Phylogenetic analysis showed that the seven cheese strains clustered together, away from the cider strain. Mating-type locus analysis revealed the presence of a MATa locus in the cider strain but a MATalpha locus in all cheese strains. The presence of LINE retrotransposons at identical genome position in the cheese strains, and two different karyotypic profiles resulting from chromosomal rearrangements were observed. Together, these findings are consistent with clonal propagation of the cheese strains. Phenotypic trait variations were observed within the cheese population under stress conditions whereas the cider strain was found to have a much greater capacity for growth in all conditions tested., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that might be considered to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Candida auris is a pathogenic yeast frequently exhibiting multidrug resistance and thus warrants special attention. The prompt detection and proper identification of this organism are needed to prevent its spread in healthcare facilities. The authors of this paper had previously developed LAMPAuris, a loop-mediated isothermal amplification assay, for the specific detection of C. auris. LAMPAuris is evaluated in this report for its ability to identify C. auris from five clades and to detect it from clinical specimens. A total of 103 skin swab samples were tested in comparison with a culture-based method and C. auris-specific SYBR green qPCR. The results show that the LAMPAuris assay had specificities ranging from 97 to 100% and sensitivities ranging from 66 to 86%. The lower sensitivity could be attributed to DNA degradation caused by the prolonged storage of the samples. In conclusion, LAMPAuris proved to be a rapid and reliable method for identifying C. auris and for detecting it in clinical specimens. Fresh specimens should ensure better yield and higher sensitivities., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

This study analyzed the prevalence and antifungal susceptibility of superficial fungal infections in 295 cases from 2019 to 2020 at a dermatology clinic. Dermatophytes were the predominant pathogens (69.5%), including Trichophytonrubrum, T. interdigitale, Microsporum canis, et al., followed by Candida spp. (29.5%), including Candidaalbicans, Ca. parapsilosis, and Ca. glabrata. The most common infections were onychomycosis (36.3%), tinea cruris (30.5%), and tinea corporis (18.6%). The distribution of SFI types showed variations based on gender, age, and season. Common antifungal agents, including terbinafine, voriconazole, ciclopiroxamine, amphotericin B, itraconazole, and ketoconazole have exhibited low minimum inhibitory concentrations against dermatophytes, especially terbinafine, which has been potent against superficial fungal infections caused by dermatophytes in the local area. Candida spp. strains were generally susceptible or classified as wild-type to 5-flucytosine and amphotericin B, with 92.0% being wild-type for itraconazole. However, resistance to fluconazole and voriconazole was observed in a small percentage of Ca. albicans and Ca. parapsilosis strains. The emergence of drug-resistant Candida underscores the importance of prudent antifungal use and continuous surveillance., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

We describe for the first time, a high-quality genome for a rare human yeast pathogen Candida mucifera, from a patient with chronic suppurative otitis media. This pathogen exhibited reduced azole susceptibility, similar to its close relatives within the Trichomonascus ciferrii species complex., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Candida species are amongst the commensals of the mucosal surfaces of the human body which include the oral cavity, vagina, and intestinal mucosa. Fungal infections are on the rise worldwide. The overall burden of infections due to fungi is difficult to estimate because the majority of them remain undiagnosed. The present study aims to determine the burden of antifungal resistance in low socioeconomic country, Pakistan and the frequency of ERG11 and MDR1 genes involved. A total of 636 Candida isolates were obtained from various tertiary care institutions in Lahore in the form of culture on various culture plates. Sabouraud agar culture plates were used to culture the Candida spp. Antifungal resistance was determined against Fluconazole, Itraconazole, Ketoconazole, and Nystatin via disk diffusion technique. Most resistance was observed against Fluconazole followed by Itraconazole, Ketoconazole, and Nystatin. The Candida isolates recovering from CVP tip and tissue have a high resistance profile. Candida species resistant to at least two antifungals were chosen for further ERG11 and MDR1 detection through real-time PCR. Among 255 Candida isolates, 240 contained ERG11 gene while MDR1 gene is present in 149 Candida isolates. The isolates carrying both genes were tested by the broth microdilution technique for the susceptibility against cycloheximide, all of them were able to grow in cycloheximide. The genetic determinants of antifungal resistance such as ERG11 and MDR1 are as important in the multidrug resistance against a variety of compounds and antifungal drugs., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The CRISPR-Cas9 system is extremely useful for genome editing in many species, including the model yeast Saccharomyces cerevisiae, and other yeast species. We have previously reported the use of an inducible CRISPR-Cas9 system in Candida glabrata, which allows genome editing but also the study of double-strand break (DSB) repair. We report, in this study, a comparable system for C. glabrata, relying on a new plasmid, which is more stable than the previous one. We also report the use of this plasmid to induce DSBs in two additional human pathogens, Candida bracarensis and Candida nivariensis. We examine lethality induced by an in vivo DSB in the three species and describe the different types of nonhomologous end-joining (NHEJ) events detected in these three pathogens., (© 2024 John Wiley & Sons Ltd.)

Background: The emerging fungal pathogen Candida auris poses a serious threat to global public health due to its worldwide distribution, multidrug resistance, high transmissibility, propensity to cause outbreaks, and high mortality. We aimed to characterise three unusual C auris isolates detected in Singapore, and to determine whether they constitute a novel clade distinct from all previously known C auris clades (I-V)., Methods: In this genotypic and phenotypic study, we characterised three C auris clinical isolates, which were cultured from epidemiologically unlinked inpatients at a large tertiary hospital in Singapore. The index isolate was detected in April, 2023. We performed whole-genome sequencing (WGS) and obtained hybrid assemblies of these C auris isolates. The complete genomes were compared with representative genomes of all known C auris clades. To provide a global context, 3651 international WGS data from the National Center for Biotechnology Information (NCBI) database were included in a high-resolution single nucleotide polymorphism (SNP) analysis. Antifungal susceptibility testing was done and antifungal resistance genes, mating-type locus, and chromosomal rearrangements were characterised from the WGS data of the three investigated isolates. We further implemented Bayesian logistic regression models to classify isolates into known clades and simulate the automatic detection of isolates belonging to novel clades as their WGS data became available., Findings: The three investigated isolates were separated by at least 37 000 SNPs (range 37 000-236 900) from all existing C auris clades. These isolates had opposite mating-type allele and different chromosomal rearrangements when compared with their closest clade IV relatives. The isolates were susceptible to all tested antifungals. Therefore, we propose that these isolates represent a new clade of C auris, clade VI. Furthermore, an independent WGS dataset from Bangladesh, accessed via the NCBI Sequence Read Archive, was found to belong to this new clade. As a proof-of-concept, our Bayesian logistic regression model was able to flag these outlier genomes as a potential new clade., Interpretation: The discovery of a new C auris clade in Singapore and Bangladesh in the Indomalayan zone, showing a close relationship to clade IV members most commonly found in South America, highlights the unknown genetic diversity and origin of C auris, particularly in under-resourced regions. Active surveillance in clinical settings, along with effective sequencing strategies and downstream analysis, will be essential in the identification of novel strains, tracking of transmission, and containment of adverse clinical effects of C auris infections., Funding: Duke-NUS Academic Medical Center Nurturing Clinician Researcher Scheme, and the Genedant-GIS Innovation Program., Competing Interests: Declaration of interests BHT declares that he serves on the advisory boards for Pfizer (for respiratory syncytial virus vaccine) and MSD (for letermovir). ALT declares that she serves on the Chapter of Pathologists, Singapore. She also received an honorarium (to her institution) for a lecture delivered to the Singapore Association of Pharmaceutical Industries. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: HG and RAF are part of the Medical Research Council Centre for Medical Mycology at University of Exeter (MR/N006364/2; MR/V033417/1). HG is supported by a Medical Research Council Doctoral Training Grant MR/P501955/2. RAF is supported by a Wellcome Trust Career Development Award (225303/Z/22/Z). JR declares no competing interests.

Candida krusei , also known as Pichia kudriavzevii , is an emerging non- albicans Candida (NAC) species causing both superficial and deep-seated infections in humans. This fungal pathogen is inherently resistant to the first-line antifungal drug, fluconazole, and is widely distributed in natural environments such as soil, foods, vegetables, and fruits. In this study, we collected 86 C. krusei strains from clinical settings and traditional fermented vegetables from different areas of China. Compared to C. krusei strains from fermented vegetables, clinical isolates exhibited a higher ability to undergo filamentation and biofilm development, which could facilitate its host colonization and infections. Isolates from fermented vegetables showed higher resistance to several antifungal drugs including fluconazole, voriconazole, itraconazole, amphotericin B, and caspofungin, than clinical strains, while they were more susceptible to posaconazole than clinical strains. Although C. krusei has been thought to be a diploid organism, we found that one-fourth of clinical strains and the majority of isolates from fermented vegetables (87.5%) are triploid. Whole-genome sequencing and population genetic analyses demonstrated that isolates from clinical settings and fermented food are genetically associated, and distributed across a wide range of genetic clusters. Additionally, we found that six nucleotide substitutions at the promoter region of the ABC11 gene, encoding a multidrug efflux pump, could play a critical role in antifungal resistance in this species. Given the ubiquitous distribution of C. krusei strains in fermented vegetables and their genetic association with clinical strains, a One Health approach will be necessary to control the prevalence of this pathogen.

Objectives: Toxicants inhibit microbial fermentation and reduce product titres. This work investigated the glycerol production characteristics of Candida glycerinogenes in highly toxic unwashed undetoxified hydrolysate and provided new ideas for high glycerol production from hydrolysates., Results: The unwashed hydrolysate contains higher concentrations of toxicants, such as furfural, acetic acid, phenols and NaCl than the washed alkali-treated bagasse hydrolysate. C. glycerinogenes fermented unwashed undetoxified hydrolysate yielded 36.1 g/L glycerol, 15.8% higher than the washed hydrolysate, suggesting that the toxicants stimulated glycerol synthesis. qRT-PCR analysis showed that toxicants of unwashed undetoxified hydrolysates greatly up-regulated the transcript levels of the genes GPD1, HXT4 and MSN4 et al. Overexpressing the above genes increased glycerol production by 27.9% to 46.1 g/L. And it was further increased by 8.8% to 50.1 g/L in a 5 L bioreactor., Conclusions: This result proves that toxicants in lignocellulosic hydrolysates can increase the titre of microbial glycerol production., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Introduction: Blood cultures have low sensitivity for candidemia. Sensitivity can be improved by the culture-independent system T2 Magnetic Resonance (T2). SeptiCyte RAPID is a host response assay quantifying the risk of infection-related inflammation through a scoring system (SeptiScore). We investigate the performance of SeptiScore in detecting persistent candidemia as defined by conventional cultures and T2., Methods: This is a prospective multicentre observational study on patients with candidemia. Blood cultures and blood samples for assessment by T2 and SeptiCyte were collected for 4 consecutive days after the index culture. The performance of SeptiScore was explored to predict persistent candidemia as defined by (1) positive follow-up blood culture (2) either positive follow-up blood culture or T2 sample., Results: 10 patients were enrolled including 34 blood collections assessed with the 3 methods. Overall, 4/34 (12%) follow-up blood cultures and 6/34 (18%) T2 samples were positive. A mixed model showed significantly higher SeptiScores associated with persistent candidemia when this was defined as either a positive follow-up blood culture or T2 sample (0.82, 95%CI 0.06 to 1.58) but not when this was defined as a positive follow-up blood culture only (-0.57, 95%CI -1.28 to 0.14). ROC curve for detection of persistent candidemia by SeptiScore at day 1 follow-up showed an AUC of 0.85 (95%CI 0.52-1.00) when candidemia was defined by positive follow-up blood culture, and an AUC of 1.00 (95%CI 1.00-1.00) when candidemia was defined according to both methods., Conclusion: Integrating transcriptome profiling with culture-independent systems and conventional cultures may increase our ability to diagnose persistent candidemia., (© 2024. The Author(s).)

Candida albicans causes millions of mucosal infections in humans annually. Hyphal overgrowth on mucosal surfaces is frequently associated with tissue damage caused by candidalysin, a secreted peptide toxin that destabilizes the plasma membrane of host cells thereby promoting disease and immunopathology. Candidalysin was first identified in C. albicans strain SC5314, but recent investigations have revealed candidalysin "variants" of differing amino acid sequence in isolates of C. albicans , and the related species C. dubliniensis , and C tropicalis , suggesting that sequence variation among candidalysins may be widespread in natural populations of these Candida species. Here, we analyzed ECE1 gene sequences from 182 C . albicans isolates, 10 C . dubliniensis isolates, and 78 C . tropicalis isolates and identified 10, 3, and 2 candidalysin variants in these species, respectively. Application of candidalysin variants to epithelial cells revealed differences in the ability to cause cellular damage, changes in metabolic activity, calcium influx, MAPK signalling, and cytokine secretion, while biophysical analyses indicated that variants exhibited differences in their ability to interact with and permeabilize a membrane. This study identifies candidalysin variants with differences in biological activity that are present in medically relevant Candida species., Importance: Fungal infections are a significant burden to health. Candidalysin is a toxin produced by Candida albicans that damages host tissues, facilitating infection. Previously, we demonstrated that candidalysins exist in the related species C. dubliniensis and C. tropicalis , thereby identifying these molecules as a toxin family. Recent genomic analyses have highlighted the presence of a small number of candidalysin "variant" toxins, which have different amino acid sequences to those originally identified. Here, we screened genome sequences of isolates of C. albicans , C. dubliniensis , and C. tropicalis and identified candidalysin variants in all three species. When applied to epithelial cells, candidalysin variants differed in their ability to cause damage, activate intracellular signaling pathways, and induce innate immune responses, while biophysical analysis revealed differences in the ability of candidalysin variants to interact with lipid bilayers. These findings suggest that intraspecies variation in candidalysin amino acid sequence may influence fungal pathogenicity., Competing Interests: The authors declare no conflict of interest.

Despite the severe impact of uncommon yeast fungal infections and the pressing need for more research on the topic, there are still few studies available on the identification, epidemiology, and susceptibility profile of those pathogens. The aims of the current study were to define the profile of uncommon yeast species at Fattouma Bourguiba University Hospital using phenotypic, molecular, and proteomic methods and to study their antifungal susceptibility profile. Pre-identified uncommon yeast species were collected from 2018 to 2021. These isolates were further identified using phenotypic methods (ID32C® system and Vitek2® YST), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and sequencing. The antifungal susceptibility profile was studied using the reference CLSI broth microdilution method. In total, 30 strains were collected during the study period. Referring to the sequencing, the most isolated uncommon species were Saprochaete capitata, Candida lusitaniae, Candida kefyr, Candida inconspicua, and Candida guilliermondii. A total of 90% of isolates were correctly identified by MALDI-TOF MS compared to 76.7% and 63.3% by ID32® C and VITEK® 2 YST, respectively. The isolated species showed variable responses to antifungals. Candida guilliermondii showed increased azole minimum inhibitory concentrations. Misidentification of uncommon yeast species was common using commercial phenotypic methods. The high percentage of concordance of MALDI-TOF results with sequencing highlights its high performance and usefulness as a routine diagnosis tool., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Objectives: Candida spp. is an opportunistic pathogen that causes superficial and invasive infections with nosocomial outbreaks without strict hygiene protocols. Herein, we assessed oral colonisation by Candida spp. in 209 Intensive Care Unit (ICU) patients between July 2021 and April 2022, conducting clinical, epidemiological, and microbiological characterisation of those developing oral or invasive candidiasis., Methods: Initial oral swabs were collected within 24 h of admission in the ICU, followed by collections on Days 2, 4, 6 and 8. Swabs from denture-wearing patients, abiotic surfaces, healthcare professionals' hands, and retroauricular regions were also obtained. Recovered yeasts and filamentous fungi were identified using MALDI-TOF MS and morphological characteristics, respectively. Genetic similarity of Candida spp. isolates was evaluated using Amplified fragment length polymorphism (AFLP), and the antifungal susceptibility profile was determined by broth microdilution., Results: In the study, 64.11% of patients were orally colonised by Candida spp. Of these, 80.59% were colonised within the first 24 h. Oral colonisation also occurred on subsequent days: 50%/Day 2, 26.92%/Day 4, and 11.53%/Days 6 and 8. Of the patients, 8.61% had oral candidiasis, mainly pseudomembranous. Among orally colonised patients, 2.23% developed invasive candidiasis. Besides, 89.47% of healthcare professionals evaluated were colonised. MALDI-TOF MS identified different yeast species, and C. albicans (45.34%), C. tropicalis (15.7%), and C. parapsilosis sensu stricto (9.88%) were the most prevalent. AFLP analysis indicated a high genetic correlation (≥97%) between C. parapsilosis sensu stricto isolates from patients and professionals. Three resistant C. albicans isolates were also found., Conclusion: This study reported a diversity of yeast and filamentous fungi species in ICU patients and highlighted early Candida spp. colonisation risks for invasive candidiasis, as well as the potential horizontal transmission in the nosocomial setting, emphasising the need for effective infection control measures., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Objectives: The investigation of Candida auris outbreaks is needed to provide insights into its population structure and transmission dynamics. We genotypically and phenotypically characterised a C. auris nosocomial outbreak occurred in Consorcio Hospital General Universitario de Valencia (CHGUV), Spain., Methods: Data and isolates were collected from CHGUV from September 2017 (first case) until September 2021. Thirty-five isolates, including one from an environmental source, were randomly selected for whole genome sequencing (WGS), and the genomes were analysed along with a database with 335 publicly available genomes, assigning them to one of the five major clades. In order to identify polymorphisms associated with drug resistance, we used the fully susceptible GCA_003014415.1 strain as reference sequence. Known mutations in genes ERG11 and FKS1 conferring resistance to fluconazole and echinocandins, respectively, were investigated. Isolates were classified into aggregating or non-aggregating., Results: All isolates belonged to clade III and were from an outbreak with a single origin. They clustered close to three publicly available genomes from a hospital from where the first patient was transferred, being the probable origin. The mutation VF125AL in the ERG11 gene, conferring resistance to fluconazole, was present in all the isolates and one isolate also carried the mutation S639Y in the FKS1 gene. All the isolates had a non-aggregating phenotype (potentially more virulent)., Conclusions: Isolates are genotypically related and phenotypically identical but one with resistance to echinocandins, which seems to indicate that they all belong to an outbreak originated from a single isolate, remaining largely invariable over the years. This result stresses the importance of implementing infection control practices as soon as the first case is detected or when a patient is transferred from a setting with known cases., (© 2024 The Author(s). Mycoses published by Wiley‐VCH GmbH.)

Meyerozyma guilliermondii (Candida guilliermondii) is one of the Candida species associated with invasive candidiasis. With the potential for expressing industrially important enzymes, M. guilliermondii strain SO possessed 99 % proteome similarity with the clinical ATCC 6260 isolate and showed pathogenicity towards zebrafish embryos. Recently, three secreted aspartyl proteinases (SAPs) were computationally identified as potential virulence factors in this strain without in vitro verification of SAP activity. The quantification of Candida SAPs activity in liquid broth were also scarcely reported. Thus, this study aimed to characterize M. guilliermondii strain SO's ability to produce SAPs (MgSAPs) in different conditions (morphology and medium) besides analyzing its growth profile. MgSAPs' capability to cleave bovine serum albumin (BSA) was also determined to propose that MgSAPs as the potential virulence factors compared to the avirulent Saccharomyces cerevisiae. M. guilliermondii strain SO produced more SAPs (higher activity) in yeast nitrogen base-BSA-dextrose broth compared to yeast extract-BSA-dextrose broth despite insignificantly different SAP activity in both planktonic and biofilm cells. FeCl 3 supplementation significantly increased the specific protein activity (∼40 %). The BSA cleavage by MgSAPs at an acidic pH was proven through semi-quantitative SDS-PAGE, sharing similar profile with HIV-1 retropepsin. The presented work highlighted the MgSAPs on fungal cell wall and extracellular milieu during host infection could be corroborated to the quantitative production in different growth modes presented herein besides shedding lights on the potential usage of retropepsin's inhibitors in treating candidiasis. Molecular and expression analyses of MgSAPs and their deletion should be further explored to attribute their respective virulence effects., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SITI NURBAYA OSLAN reports financial support was provided by Malaysia Ministry of Higher Education., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The rise of Candida auris , a multidrug-resistant fungal pathogen, across more than 40 countries, has signaled an alarming threat to global health due to its significant resistance to existing antifungal therapies. Characterized by its rapid spread and robust drug resistance, C. auris presents a critical challenge in managing infections, particularly in healthcare settings. With research on its biological traits and genetic basis of virulence and resistance still in the early stages, there is a pressing need for a concerted effort to understand and counteract this pathogen. This review synthesizes current knowledge on the epidemiology, biology, genetic manipulation, pathogenicity, diagnostics, and resistance mechanisms of C. auris , and discusses future directions in research and therapeutic development. By exploring the complexities surrounding C. auris , we aim to underscore the importance of advancing research to devise effective control and treatment strategies.

Candida auris is a multidrug-resistant fungal pathogen with a propensity to colonize humans and persist on environmental surfaces. C. auris invasive fungal disease is being increasingly identified in acute and long-term care settings. We have developed a prototype cartridge-based C. auris surveillance assay (CaurisSurV cartridge; "research use only") that includes integrated sample processing and nucleic acid amplification to detect C. auris from surveillance skin swabs in the GeneXpert instrument and is designed for point-of-care use. The assay limit of detection (LoD) in the skin swab matrix was 10.5 and 14.8 CFU/mL for non-aggregative (AR0388) and aggregative (AR0382) strains of C. auris , respectively. All five known clades of C. auris were detected at 2-3-5× (31.5-52.5 CFU/mL) the LoD. The assay was validated using a total of 85 clinical swab samples banked at two different institutions (University of California Los Angeles, CA and Wadsworth Center, NY). Compared to culture, sensitivity was 96.8% (30/31) and 100% (10/10) in the UCLA and Wadsworth cohorts, respectively, providing a combined sensitivity of 97.5% (40/41), and compared to PCR, the combined sensitivity was 92% (46/50). Specificity was 100% with both clinical ( C. auris negative matrix, N = 31) and analytical (non- C . auris strains, N = 32) samples. An additional blinded study with N = 60 samples from Wadsworth Center, NY yielded 97% (29/30) sensitivity and 100% (28/28) specificity. We have developed a completely integrated, sensitive, specific, and 58-min prototype test, which can be used for routine surveillance of C. auris and might help prevent colonization and outbreaks in acute and chronic healthcare settings., Importance: This study has the potential to offer a better solution to healthcare providers at hospitals and long-term care facilities in their ongoing efforts for effective and timely control of Candida auris infection and hence quicker response for any potential future outbreaks., Competing Interests: D.A. receives research support and royalty payments from Cepheid. B.O. and S.C. are employees of Cepheid. The other authors do not declare any competing interests.

Candida auris is an emerging fungal pathogen associated with multi-drug resistance rates and widespread outbreaks in hospitals and healthcare 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 2 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., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Candida lusitaniae fungemia is a serious infection that is rarely reported in children. The aim of this study is to describe a case series of C. lusitaniae fungemia and review previous publications regarding this rare pathogen. This is a multicenter case series of children diagnosed with C. lusitaniae fungemia. A total of 18 cases that occurred over a 15-year period in five tertiary hospitals were included. Additionally, a review of the literature regarding C. lusitaniae fungemia in children was performed. A total of 18 cases were enrolled; 11/18 (61%) were males, with a mean age of 2.3 years. All patients had severe underlying diseases and risk factors for opportunistic infection, most commonly prematurity and malignancies. More than one-third of cases occurred during the last 2 years of the study period. All isolates were susceptible to all tested antifungals. The survival rate following the acute infection was 94%, whereas the survival rate of 14 previously published cases was 71%, with the most common underlying diseases being CGD and malignancies. Candida lusitaniae fungemia is not a common event in the pediatric population, occurring exclusively in children with severe underlying diseases and significant risk factors. This cohort revealed better clinical outcomes than previously reported. All tested isolates were susceptible to all antifungal agents; variability in susceptibility as previously reported was not found in this study. The allegedly higher rate of infection in recent years is in need of further investigation in larger prospective studies in order to conclude if a real trend is at play., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Candida auris represents one of the most urgent threats to public health, although its ecology remains largely unknown. Because amphibians and reptiles may present favorable conditions for C. auris colonization, cloacal and blood samples (n = 68), from several snake species, were cultured and molecularly screened for C. auris using molecular amplification of glycosylphosphatidylinositol protein-encoding genes and ribosomal internal transcribed spacer sequencing. Candida auris was isolated from the cloacal swab of one Egyptian cobra (Naja haje legionis) and molecularly identified in its cloaca and blood. The isolation of C. auris from wild animals is herein reported for the first time, thus suggesting the role that these animals could play as reservoirs of this emerging pathogen. The occurrence of C. auris in blood requires further investigation, although the presence of cationic antimicrobial peptides in the plasma of reptiles could play a role in reducing the vitality of the fungus., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Candida auris , initially identified in 2009, has rapidly become a critical concern due to its antifungal resistance and significant mortality rates in healthcare-associated outbreaks. To date, whole-genome sequencing (WGS) has identified five unique clades of C. auris , with some strains displaying resistance to all primary antifungal drug classes. In this study, we presented the first WGS analysis of C. auris from Bangladesh, describing its origins, transmission dynamics, and antifungal susceptibility testing (AFST) profile. Ten C. auris isolates collected from hospital settings in Bangladesh were initially identified by CHROMagar Candida Plus, followed by VITEK2 system, and later sequenced using Illumina NextSeq 550 system. Reference-based phylogenetic analysis and variant calling pipelines were used to classify the isolates in different clades. All isolates aligned ~90% with the Clade I C. auris B11205 reference genome. Of the 10 isolates, 8 were clustered with Clade I isolates, highlighting a South Asian lineage prevalent in Bangladesh. Remarkably, the remaining two isolates formed a distinct cluster, exhibiting >42,447 single-nucleotide polymorphism differences compared to their closest Clade IV counterparts. This significant variation corroborates the emergence of a sixth clade (Clade VI) of C. auris in Bangladesh, with potential for international transmission. AFST results showed that 80% of the C. auris isolates were resistant to fluconazole and voriconazole, whereas Clade VI isolates were susceptible to azoles, echinocandins, and pyrimidine analogue. Genomic sequencing revealed ERG11 _Y132F mutation conferring azole resistance while FCY1 _S70R mutation found inconsequential in describing 5-flucytosine resistance. Our study underscores the pressing need for comprehensive genomic surveillance in Bangladesh to better understand the emergence, transmission dynamics, and resistance profiles of C. auris infections. Unveiling the discovery of a sixth clade (Clade VI) accentuates the indispensable role of advanced sequencing methodologies.IMPORTANCE Candida auris is a nosocomial fungal pathogen that is commonly misidentified as other Candida species. Since its emergence in 2009, this multidrug-resistant fungus has become one of the five urgent antimicrobial threats by 2019. Whole-genome sequencing (WGS) has proven to be the most accurate identification technique of C. auris which also played a crucial role in the initial discovery of this pathogen. WGS analysis of C. auris has revealed five distinct clades where isolates of each clade differ among themselves based on pathogenicity, colonization, infection mechanism, as well as other phenotypic characteristics. In Bangladesh, C. auris was first reported in 2019 from clinical samples of a large hospital in Dhaka city. To understand the origin, transmission dynamics, and antifungal-resistance profile of C. auris isolates circulating in Bangladesh, we conducted a WGS-based surveillance study on two of the largest hospital settings in Dhaka, Bangladesh., Competing Interests: The authors declare no conflict of interest.

Background: Candida vulturna is an emerging pathogen belonging to the Metshnikowiaceae family together with Candida auris and Candida haemulonii species complex. Some strains of this species were reported to be resistant to several antifungal agents., Objectives: This study aims to address identification difficulties, evaluate antiungal susceptibilities and explore the molecular mechanisms of azole resistance of Candida vulturna., Methods: We studied five C. vulturna clinical strains isolated in three Colombian cities. Identification was performed by phenotypical, proteomic and molecular methods. Antifungal susceptibility testing was performed following CLSI protocol. Its ERG11 genes were sequenced and a substitution was encountered in azole resistant isolates. To confirm the role of this substitution in the resistance phenotype, Saccharomyces cerevisiae strains with a chimeric ERG11 gene were created., Results: Discrepancies in identification methods are highlighted. Sequencing confirmed the identification as C. vulturna. Antifungal susceptibility varied among strains, with four strains exhibiting reduced susceptibility to azoles and amphotericin B. ERG11 sequencing showed a point mutation (producing a P135S substitution) that was associated with the azole-resistant phenotype., Conclusions: This study contributes to the understanding of C. vulturna's identification challenges, its susceptibility patterns, and sheds light on its molecular mechanisms of azole resistance., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Fungal diseases are often linked to poverty, which is associated with poor hygiene and sanitation conditions that have been severely worsened by the COVID-19 pandemic. Moreover, COVID-19 patients are treated with Dexamethasone, a corticosteroid that promotes an immunosuppressive profile, making patients more susceptible to opportunistic fungal infections, such as those caused by Candida species. In this study, we analyzed the prevalence of Candida yeasts in wastewater samples collected to track viral genetic material during the COVID-19 pandemic and identified the yeasts using polyphasic taxonomy. Furthermore, we investigated the production of biofilm and hydrolytic enzymes, which are known virulence factors. Our findings revealed that all Candida species could form biofilms and exhibited moderate hydrolytic enzyme activity. We also proposed a workflow for monitoring wastewater using Colony PCR instead of conventional PCR, as this technique is fast, cost-effective, and reliable. This approach enhances the accurate taxonomic identification of yeasts in environmental samples, contributing to environmental monitoring as part of the One Health approach, which preconizes the monitoring of possible emergent pathogenic microorganisms, including fungi., (© 2024. The Author(s).)

NAD is a redox coenzyme and is the center of energy metabolism. In metabolic engineering modifications, an insufficient NAD(H) supply often limits the accumulation of target products. In this study, Candida glycerinogenes was found to be able to supply NAD(H) in large fluxes, up to 7.6 times more than Saccharomyces cerevisiae in aerobic fermentation. Aerobic fermentation in a medium without amino nitrogen sources demonstrated that C. glycerinogenes NAD synthesis was not dependent on NAD precursors in the medium. Inhibition by antisense RNA and the detection of transcript levels indicated that the main NAD supply pathway is the de novo biosynthesis pathway. It was further demonstrated that NAD(H) supply was unaffected by changes in metabolic flow through C. glycerinogenes Δ GPD aerobic fermentation (80 g/L ethanol). In conclusion, the ability of C. glycerinogenes to supply NAD(H) in large fluxes provides a new approach to solving the NAD(H) supply problem in synthetic biology.

Candida auris is a multidrug-resistant opportunistic fungal pathogen capable of causing serious infections and healthcare-associated outbreaks. Screening for colonization with C. auris has become routine and is recommended in many hospitals and healthcare facilities as an infection control and prevention strategy. Subsequently, and since there are currently no FDA-approved tests for this purpose, clinical microbiology laboratories have become responsible for developing protocols to detect C. auris using axial and inguinal screening swabs. In a College of American Pathologists-accredited large academic healthcare center setting, we implemented a laboratory-developed nucleic-acid amplification test for the detection of C. auris DNA. Our test validation evaluated the performance of the DiaSorin C. auris primer set used in a real-time qualitative PCR assay on the LIAISON MDX thermocycler with the Simplexa Universal Disc. The assay was highly sensitive and specific, with a limit of detection of 1-2 CFU/reaction, with no observed cross-reactivity with other Candida spp., bacterial skin commensal organisms or commonly encountered viruses. When run in parallel with a culture-based detection method, the PCR assay was 100% sensitive and specific. The assay was precise, with low variability between replicates within and between runs. Lastly, pre-analytical factors, including swab storage time, temperature, and transport media, were assessed and found to have no significant effect on the detection of C. auris at variable concentrations. Taken together, this study expands the available options for nucleic acid detection of C. auris and characterizes pre-analytical factors for implementation in both high- and low-volume laboratory settings., Importance: This study overviews the validation and implementation of a molecular screening tool for the detection of Candida auris in a College of American Pathologist-accredited clinical laboratory. This molecular laboratory-developed test is both highly sensitive and specific and has significant health-system cost-savings associated with significantly reduced turn-around-time compared to traditional standard-of-care culture-based work up. This method and workflow is of interest to support clinical microbiology diagnostics and to help aid in hospital inpatient, and infection prevention control screening., Competing Interests: The authors declare no conflict of interest.

Fungal infections in neonatal intensive care units (NICU) are mainly related to Candida species, with high mortality rates. They are predominantly of endogenous origin, however, cross-infection transmitted by healthcare professionals' hands has occurred. The aim of this study was to identify Candida species isolated from the hands of healthcare professionals in a NICU before and after hygiene with 70% ethanol-based gel and evaluate virulence factors DNase, phospholipase, proteinase, hemolysin, biofilm biomass production, and metabolic activity. In vitro antifungal susceptibility testing and similarity by random amplified polymorphic DNA (RAPD) were also performed. C. parapsilosis complex was the most frequent species (57.1%); all isolates presented at least one virulence factor; three isolates (Candida parapsilosis complex) were resistant to amphotericin B, two (Candida famata [currently Debaryomyces hansenii] and Candida guilliermondii [currently Meyerozyma guilliermondii]) was resistant to micafungin, and six (Candida parapsilosis complex, Candida guilliermondii [=Meyerozyma guilliermondii], Candida viswanathi, Candida catenulata [currently Diutina catenulata] and Candida lusitaniae [currently Clavispora lusitaniae]) were resistant to fluconazole. Molecular analysis by RAPD revealed two clusters of identical strains that were in the hands of distinct professionals. Candida spp. were isolated even after hygiene with 70% ethanol-based gel, highlighting the importance of stricter basic measures for hospital infection control to prevent nosocomial transmission., Competing Interests: Declaration of competing interest none., (Copyright © 2024. Published by Elsevier Masson SAS.)