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Candida parapsilosis is one of the most commen causes of life-threatening candidaemia, particularly in premature neonates, individuals with cancer of the haematopoietic system, and recipients of organ transplants. Historically, drug-susceptible strains have been linked to clonal outbreaks. However, worldwide studies started since 2018 have reported severe outbreaks among adults caused by fluconazole-resistant strains. Outbreaks caused by fluconazole-resistant strains are associated with high mortality rates and can persist despite strict infection control strategies. The emergence of resistance threatens the efficacy of azoles, which is the most widely used class of antifungals and the only available oral treatment option for candidaemia. The fact that most patients infected with fluconazole-resistant strains are azole-naive underscores the high potential adaptability of fluconazole-resistant strains to diverse hosts, environmental niches, and reservoirs. Another concern is the multidrug-resistant and echinocandin-tolerant C parapsilosis isolates, which emerged in 2020. Raising awareness, establishing effective clinical interventions, and understanding the biology and pathogenesis of fluconazole-resistant C parapsilosis are urgently needed to improve treatment strategies and outcomes., Competing Interests: Declaration of interests CJN is a cofounder of BioSynesis, a company developing diagnostics and therapeutics for biofilm infections. MH reports grants and research funding from Astellas Pharma, Gilead Sciences, MSD, Pfizer, Euroimmun, F2G, Pulmocide, IMMY, Mundipharma, and Scynexis, outside the submitted work. TG acknowledges support from the Spanish Ministry of Science and Innovation (PID2021–126067NB-I00), cofounded by European Regional Development Fund, the Catalan Research Agency (AGAUR) SGR423, the European Union's Horizon 2020 research and innovation programme (ERC-2016–724173); the Gordon and Betty Moore Foundation (GBMF9742), the La Caixa foundation (LCF/PR/HR21/00737), and the Instituto de Salud Carlos III (IMPACT Grant IMP/00019, and CIBERINFEC CB21/13/00061- ISCIII-SGEFI/ERDF). BZ acknowledges support from the National Key Research and Development Program of China 2021YFA0911300. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Patients presenting with severe COVID-19 are predisposed to acquire secondary fungal infections such as COVID-19-associated candidemia (CAC), which are associated with poor clinical outcomes despite antifungal treatment. The extreme burden imposed on clinical facilities during the COVID-19 pandemic has provided a permissive environment for the emergence of clonal outbreaks of multiple Candida species, including C. auris and C. parapsilosis . Here we report the largest clonal CAC outbreak to date caused by fluconazole resistant (FLZR) and echinocandin tolerant (ECT) C. parapsilosis . Sixty C. parapsilosis strains were obtained from 57 patients at a tertiary care hospital in Brazil, 90% of them were FLZR and ECT. Although only 35.8% of FLZR isolates contained an ERG11 mutation, all of them contained the TAC1 L518F mutation and significantly overexpressed CDR1 . Introduction of TAC1 L518F into a susceptible background increased the MIC of fluconazole and voriconazole 8-fold and resulted in significant basal overexpression of CDR1 . Additionally, FLZR isolates exclusively harboured E1939G outside of Fks1 hotspot-2, which did not confer echinocandin resistance, but significantly increased ECT. Multilocus microsatellite typing showed that 51/60 (85%) of the FLZR isolates belonged to the same cluster, while the susceptible isolates each represented a distinct lineage. Finally, biofilm production in FLZR isolates was significantly lower than in susceptible counterparts Suggesting that it may not be an outbreak determinant. In summary, we show that TAC1 L518F and FKS1 E1393G confer FLZR and ECT, respectively, in CAC-associated C. parapsilosis . Our study underscores the importance of antifungal stewardship and effective infection control strategies to mitigate clonal C. parapsilosis outbreaks.

Fluconazole-resistant Candida parapsilosis (FLZR-CP) outbreaks are a growing public health concern and have been reported in numerous countries. Patients infected with FLZR-CP isolates show fluconazole therapeutic failure and have a significantly increased mortality rate. Because fluconazole is the most widely used antifungal agent in most regions with outbreaks, it is paramount to restore its antifungal activity. Milbemycin oxim (MOX), a well-known canine endectocide, is a potent efflux pump inhibitor that significantly potentiates the activity of fluconazole against FLZR C . glabrata and C . albicans. However, the FLZ-MOX combination has not been tested against FLZR-CP isolates, nor is it known whether MOX may also potentiate the activity of echinocandins, a different class of antifungal drugs. Furthermore, the extent of involvement of efflux pumps CDR1 and MDR1 and ergosterol biosynthesis enzyme ERG11 and their link with gain-of-function (GOF) mutations in their transcription regulators ( TAC1 , MRR1 , and UPC2 ) are poorly characterized among FLZR-CP isolates. We analyzed 25 C. parapsilosis isolates collected from outbreaks in Turkey and Brazil by determining the expression levels of CDR1 , MDR1 , and ERG11 , examining the presence of potential GOF mutations in their transcriptional regulators, and assessing the antifungal activity of FLZ-MOX and micafungin-MOX against FLZR and multidrug-resistant (MDR) C . parapsilosis isolates. ERG11 was found to be universally induced by fluconazole in all isolates, while expression of MDR1 was unchanged. Whereas mutations in MRR1 and UPC2 were not detected, CDR1 was overexpressed in three Brazilian FLZR-CP isolates, which also carried a novel TAC1 L518F mutation. Of these three isolates, one showed increased basal expression of CDR1 , while the other two overexpressed CDR1 only in the presence of fluconazole. Interestingly, MOX showed promising antifungal activity against FLZR isolates, reducing the FLZ MIC 8- to 32-fold. However, the MOX and micafungin combination did not exert activity against an MDR C . parapsilosis isolate. Collectively, our study documents that the mechanisms underpinning FLZR are region specific, where ERG11 mutations were the sole mechanism of FLZR in Turkish FLZR-CP isolates, while simultaneous overexpression of CDR1 was observed in some Brazilian counterparts. Moreover, MOX and fluconazole showed potent synergistic activity, while the MOX-micafungin combination showed no synergy., Competing Interests: AC received educational Grants from Angem, Eurofarma, Knight-United Medical, Gilead, Pfizer and travel grant support from Eurofarma and Knight-United Medical. DP receives research support and/or serves on advisory boards for Amplyx, Cidara, Scynexis, N8 Medical, Merck, Regeneron, and Pfizer. He also has a patent covering the detection of fungal species and drug resistance, as well as a pending patent on COVID-19 detection licensed to T2 Biosystems. The remaining 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 © 2022 Daneshnia, Hilmioğlu Polat, Ilkit, Shor, de Almeida Júnior, Favarello, Colombo, Arastehfar and Perlin.)

Species of Trichosporon and related genera are widely used in biotechnology and, hence, many species have their genome sequenced. Importantly, yeasts of the genus Trichosporon have been increasingly identified as a cause of life-threatening invasive trichosporonosis (IT) in humans and are associated with an exceptionally high mortality rate. Trichosporon spp. are intrinsically resistant to frontline antifungal agents, which accounts for numerous reports of therapeutic failure when echinocandins are used to treat IT. Moreover, these fungi have low sensitivity to polyenes and azoles and, therefore, are potentially regarded as multidrug-resistant pathogens. However, despite the clinical importance of Trichosporon spp., our understanding of their antifungal resistance mechanisms is quite limited. Furthermore, antifungal susceptibility testing is not standardized, and there is a lack of interpretive epidemiological cut-off values for minimal inhibitory concentrations to distinguish non-wild type Trichosporon isolates. The route of infection remains obscure and detailed clinical and environmental studies are required to determine whether the Trichosporon infections are endogenous or exogenous in nature. Although our knowledge on effective IT treatments is rather limited and future randomized clinical trials are required to identify the best antifungal agent, the current paradigm advocates the use of voriconazole, removal of central venous catheters and recovery from neutropenia.

Candida parapsilosis produces biofilm, which colonizes catheters and other invasive medical devices that are manipulated by health care workers. In previous studies, C. parapsilosis in vitro biofilms have exhibited high resistance rates against conventional antifungals, but susceptibility to both echinocandins and lipid formulations of amphotericin B (lipid complex and liposomal). However, a recent study showed good activity of amphotericin B deoxycholate on the biomass of C. parapsilosis biofilms. Although moderate activity of echinocandins has been demonstrated against low metabolic activity biofilms of C. parapsilosis, few studies have analyzed the action of these drugs on high metabolic activity biofilms. Moreover, high biofilm-forming isolates have been associated with central venous catheter-related fungemia outbreaks and higher mortality rates. Therefore, it is relevant to verify the activity of the main antifungal drugs against high metabolic activity biofilms of C. parapsilosis. Our study aimed to evaluate the in vitro activity of amphotericin B deoxycholate, anidulafungin, caspofungin, and micafungin against high biofilm-forming and high metabolic activity clinical isolates of C. parapsilosis. Our results showed good activity of amphotericin B against C. parapsilosis biofilms, but none of the echinocandin drugs was effective. This suggests that amphotericin B deoxycholate may be a better choice than echinocandins for the treatment of biofilm-associated infections by C. parapsilosis, mainly in countries with insufficient health care resources to purchase lipid formulations of amphotericin B. These results warn of the possibility of persistent catheter-related candidemia caused by high biofilm-forming C. parapsilosis strains when treated with echinocandin drugs.

There is worldwide concern with the increasing rates of infections due to multiresistant Candida isolates reported in tertiary medical centers. We checked for historical trends in terms of prevalence rates and antifungal susceptibility of the Candida haemulonii species complex in our yeast stock culture collected during the last 11 years. The isolates were identified by sequencing the rDNA internal transcribed spacer (ITS) region, and antifungal susceptibility tests for amphotericin B, voriconazole, fluconazole, anidulafungin, and 5-fluorocytosine were performed by the Clinical and Laboratory Standards Institute (CLSI) microbroth method. A total of 49 isolates were identified as Candida haemulonii sensu stricto ( n = 21), followed by C. haemulonii var. vulnera ( n = 15) and C. duobushaemulonii ( n = 13), including 38 isolates cultured from patients with deep-seated Candida infections. The prevalence of the C. haemulonii species complex increased from 0.9% (18 isolates among 1931) in the first period (December 2008 to June 2013) to 1.7% (31 isolates among 1868) in the second period (July 2014 to December 2019) of analysis ( p = 0.047). All isolates tested exhibited high minimum inhibition concentrations for amphotericin B and fluconazole, but they remained susceptible to 5-fluorocytosine and anidulafungin. We were able to demonstrate the increased isolation of the multiresistant Candida haemulonii species complex in our culture collection, where most isolates were cultured from patients with deep-seated infections.

Critically ill patients and patients with haematological cancer are HIV-negative populations at high risk of invasive fungal infections. In intensive-care units, candidaemia and intra-abdominal candidiasis predominate, but aspergillosis has emerged as a lethal, under-recognised cause of pneumonia. In patients with haematological malignancies or who have undergone stem-cell transplantations, pulmonary disease due to aspergillus and other mould diseases predominate. In this Series paper, we provide an update on risk assessment, new diagnostic strategies, and therapeutic approaches. New concepts have emerged for use of risk prediction rules and an evidence base now exists for inclusion of biomarkers (eg, galactomannan, 1,3-β-D-glucan, and PCR assays for Aspergillus spp) into early diagnostic and therapeutic strategies. Imaging techniques remain helpful for early diagnosis of pulmonary mould diseases, with PET techniques offering potential improvements in diagnostic specificity and evaluation of clinical response. Echinocandins and triazoles have been validated extensively for prophylaxis, empirical therapy, and targeted therapy, but an increase in intrinsically resistant fungi and emergence of secondary resistance as a result of drug-induced selection pressure are of major concern. Echinocandins remain a major component of treatment of invasive candidiasis and new triazoles are the best alternative for prophylaxis and therapy of invasive aspergillosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: This review aimed to better depict the clinical features and address the issue of therapeutic management of Trichosporon deep-seated infections. Methods: We comprehensively reviewed the cases of invasive Trichosporon infection reported in the literature from 1994 (date of taxonomic modification) to 2015. Data from antifungal susceptibility testing (AST) studies were also analyzed. Results: Two hundred and three cases were retained and split into four groups: homeopathy ( n = 79), other immunodeficiency conditions ( n = 41), miscellaneous ( n = 58) and newborns ( n = 25). Trichosporon asahii was the main causative species (46.7%) and may exhibit cross-resistance to different antifungal classes. The unfavorable outcome rate was at 44.3%. By multivariate analysis, breakthrough infection (OR 2.45) was associated with unfavorable outcome, whilst the use of an azole-based therapy improved the prognosis (OR 0.16). Voriconazole-based treatment was associated with favorable outcome in hematological patients (73.6 vs. 41.8%; p = 0.016). Compiled data from AST demonstrated that (i) T. asahii exhibits the highest MICs to amphotericin B and (ii) voriconazole has the best in vitro efficacy against clinical isolates of Trichosporon spp. Conclusions: Trichosporon infection is not only restricted to hematological patients. Analysis of compiled data from AST and clinical outcome support the use of voriconazole as first line therapy.

Candida haemulonii is now considered a complex of two species and one variety: C. haemulonii sensu stricto, Candida duobushaemulonii and the variety C. haemulonii var. vulnera. Identification (ID) of these species is relevant for epidemiological purposes and for therapeutic management, but the different phenotypic commercial systems are unable to provide correct species ID for these emergent pathogens. Hence, we evaluated the MALDI-TOF MS performance for the ID of C. haemulonii species, analyzing isolates/strains of C. haemulonii complex species, Candida pseudohaemulonii and Candida auris by two commercial platforms, their databases and softwares. To differentiate C. haemulonii sensu sctricto from the variety vulnera, we used the ClinProTools(TM) models and a single-peak analysis with the software FlexAnalysis(TM). The Biotyper(TM) database gave 100% correct species ID for C. haemulonii sensu stricto, C. pseudohaemulonii and C. auris, with 69% of correct species ID for C. duobushaemulonii. Vitek MS(TM) IVD database gave 100% correct species ID for C. haemulonii sensu stricto, misidentifying all C. duobushaemulonii and C. pseudohaemulonii as C. haemulonii, being unable to identify C. auris. The Vitek MS(TM) RUO database needed to be upgraded with in-house SuperSpectra to discriminate C. haemulonii sensu stricto, C. duobushaemulonii, C. pseudohaemulonii, and C. auris strains/isolates. The generic algorithm model from ClinProTools(TM) software showed recognition capability of 100% and cross validation of 98.02% for the discrimination of C. haemulonii sensu stricto from the variety vulnera. Single-peak analysis showed that the peaks 5670, 6878, or 13750 m/z can distinguish C. haemulonii sensu stricto from the variety vulnera.

Neutropenic patients are at risk of the development of hyalohyphomycosis and mucormycosis. Correct identification is essential for the initiation of the specific treatment, but concomitant mold infections are rarely reported. We report one unprecedented case of concomitant mucormycosis and fusariosis in a neutropenic patient with acute myeloid leukemia. The patient developed rhino-orbital infection by Rhizopus arrhizus and disseminated infection by Fusarium solani. The first culture from a sinus biopsy grew Rhizopus, which was consistent with the histopathology report of mucormycosis. A second sinus biopsy collected later during the patient's clinical deterioration was reported as hyalohyphomycosis, and the culture yielded F. solani. Due to the discordant reports, the second biopsy was reviewed and two hyphae types suggestive of both hyalohyphomycetes and mucormycetes were found. The dual mold infection was confirmed by PCR assays from paraffinized tissue sections. Increased awareness of the existence of dual mold infections in at-risk patients is necessary. PCR methods in tissue sections may increase the diagnosis of dual mold infections. In case of sequential biopsies showing discrepant results, mixed infections have to be suspected.

We described the impact of the capsule size for Cryptococcus neoformans and Cryptococcus gattii identification at the species level by Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). After experimental capsule size modulation, we observed that reducing the capsule size resulted in improved identification by Bruker MALDI-TOF MS across all of the reference strains analyzed., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Trichosporon spp. have recently emerged as significant human pathogens. Identification of these species is important, both for epidemiological purposes and for therapeutic management, but conventional identification based on biochemical traits is hindered by the lack of updates to the species databases provided by the different commercial systems. In this study, 93 strains, or isolates, belonging to 16 Trichosporon species were subjected to both molecular identification using IGS1 gene sequencing and matrix-assisted laser desorption ionisation-time-of-flight (MALDI-TOF) analysis. Our results confirmed the limits of biochemical systems for identifying Trichosporon species, because only 27 (36%) of the isolates were correctly identified using them. Different protein extraction procedures were evaluated, revealing that incubation for 30 min with 70% formic acid yields the spectra with the highest scores. Among the six different reference spectra databases that were tested, a specific one composed of 18 reference strains plus seven clinical isolates allowed the correct identification of 67 of the 68 clinical isolates (98.5%). Although until recently it has been less widely applied to the basidiomycetous fungi, MALDI-TOF appears to be a valuable tool for identifying clinical Trichosporon isolates at the species level., (© 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.)