Echinocandin persistence directly impacts the evolution of resistance and survival of the pathogenic fungus Candida glabrata.

Recent epidemiological studies documented an alarming increase in the prevalence of echinocandin-resistant (ECR) Candida glabrata blood isolates. ECR isolates are known to arise from a minor subpopulation of a clonal population, termed echinocandin persisters. Although it is believed that isolates with a higher echinocandin persistence (ECP) are more likely to develop ECR, the implication of ECP needs to be better understood. Moreover, replacing laborious and time-consuming traditional approaches to determine ECP levels with rapid, convenient, and reliable tools is imperative to advance our understanding of this emerging concept in clinical practice. Herein, using extensive ex vivo and in vivo systemic infection models, we showed that high ECP isolates are less effectively cleared by micafungin treatment and exclusively give rise to ECR colonies. Additionally, we developed a flow cytometry-based tool that takes advantage of a SYTOX-based assay for the stratification of ECP levels. Once challenged with various collections of echinocandin-susceptible blood isolates, our assay reliably differentiated ECP levels in vitro and predicted ECP levels in real time under ex vivo and in vivo conditions when compared to traditional methods relying on colony-forming unit counting. Given the high and low ECP predictive values of 92.3% and 82.3%, respectively, our assay showed a high agreement with traditional approach. Collectively, our study supports the concept of ECP level determination in clinical settings and provides a robust tool scalable for high-throughput settings. Application of this tool facilitates the interrogation of mutant and drug libraries to further our understanding of persister biology and designing anti-persister therapeutics. IMPORTANCE: Candida glabrata is a prevalent fungal pathogen able to replicate inside macrophages and rapidly develop resistance against frontline antifungal echinocandins. Multiple studies have shown that echinocandin resistance is fueled by the survival of a small subpopulation of susceptible cells surviving lethal concentrations of echinocandins. Importantly, bacterial pathogens that exhibit high antibiotic persistence also impose a high burden and generate more antibiotic-resistant colonies. Nonetheless, the implications of echinocandin persistence (ECP) among the clinical isolates of C. glabrata have not been defined. Additionally, ECP level determination relies on a laborious and time-consuming method, which is prone to high variation. By exploiting in vivo systemic infection and ex vivo models, we showed that C. glabrata isolates with a higher ECP are associated with a higher burden and more likely develop echinocandin resistance upon micafungin treatment. Additionally, we developed an assay that reliably determines ECP levels in real time. Therefore, our study identified C. glabrata isolates displaying high ECP levels as important entities and provided a reliable and convenient tool for measuring echinocandin persistence, which is extendable to other fungal and bacterial pathogens.

Evaluation of outbreak persistence caused by multidrug-resistant and echinocandin-resistant Candida parapsilosis using multidimensional experimental and epidemiological approaches.

Candida parapsilosis is known to cause severe and persistent outbreaks in clinical settings. Patients infected with multidrug-resistant C. parapsilosis (MDR Cp) isolates were identified in a large Turkish hospital from 2017-2020. We subsequently identified three additional patients infected with MDR Cp isolates in 2022 from the same hospital and two echinocandin-resistant (ECR) isolates from a single patient in another hospital. The increasing number of MDR and ECR isolates contradicts the general principle that the severe fitness cost associated with these phenotypes could prevent their dominance in clinical settings. Here, we employed a multidimensional approach to systematically assess the fitness costs of MDR and ECR C. parapsilosis isolates. Whole-genome sequencing revealed a novel MDR genotype infecting two patients in 2022. Despite severe in vitro defects, the levels and tolerances of the biofilms of our ECR and MDR isolates were generally comparable to those of susceptible wild-type isolates. Surprisingly, the MDR and ECR isolates showed major alterations in their cell wall components, and some of the MDR isolates consistently displayed increased tolerance to the fungicidal activities of primary human neutrophils and were more immunoevasive during exposure to primary human macrophages. Our systemic infection mouse model showed that MDR and ECR C. parapsilosis isolates had comparable fungal burden in most organs relative to susceptible isolates. Overall, we observed a notable increase in the genotypic diversity and frequency of MDR isolates and identified MDR and ECR isolates potentially capable of causing persistent outbreaks in the future.

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

IMPORTANCE: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite." This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omics technologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and, therefore, are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, which can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

Small colony variants (SCVs) are relatively common among some bacterial species and are associated with poor prognosis and recalcitrant infections. Similarly, Candida glabrata - a major intracellular fungal pathogen - produces small and slow-growing respiratory-deficient colonies, termed "petite." Despite reports of clinical petite C . glabrata strains, our understanding of petite behavior in the host remains obscure. Moreover, controversies exist regarding in-host petite fitness and its clinical relevance. Herein, we employed whole-genome sequencing (WGS), dual-RNAseq, and extensive ex vivo and in vivo studies to fill this knowledge gap. WGS identified multiple petite-specific mutations in nuclear and mitochondrially-encoded genes. Consistent with dual-RNAseq data, petite C . glabrata cells did not replicate inside host macrophages and were outcompeted by their non-petite parents in macrophages and in gut colonization and systemic infection mouse models. The intracellular petites showed hallmarks of drug tolerance and were relatively insensitive to the fungicidal activity of echinocandin drugs. Petite-infected macrophages exhibited a pro-inflammatory and type I IFN-skewed transcriptional program. Interrogation of international C . glabrata blood isolates ( n =1000) showed that petite prevalence varies by country, albeit at an overall low prevalence (0-3.5%). Collectively, our study sheds new light on the genetic basis, drug susceptibility, clinical prevalence, and host-pathogen responses of a clinically overlooked phenotype in a major fungal pathogen. Importance: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite". This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omicstechnologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and therefore are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex-vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Whole-genome sequencing confirms a persistent candidaemia clonal outbreak due to multidrug-resistant Candida parapsilosis.

OBJECTIVES: Although perceived as a rare clinical entity, recent studies have noted the emergence of MDR C. parapsilosis (MDR-Cp) isolates from single patients (resistant to both azole and echinocandins). We previously reported a case series of MDR-Cp isolates carrying a novel FKS1R658G mutation. Herein, we identified an echinocandin-naive patient infected with MDR-Cp a few months after the previously described isolates. WGS and CRISPR-Cas9 editing were used to explore the origin of the new MDR-Cp isolates, and to determine if the novel mutation confers echinocandin resistance. METHODS: WGS was applied to assess the clonality of these isolates and CRISPR-Cas9 editing and a Galleria mellonella model were used to examine whether FKS1R658G confers echinocandin resistance. RESULTS: Fluconazole treatment failed, and the patient was successfully treated with liposomal amphotericin B (LAMB). WGS proved that all historical and novel MDR-Cp strains were clonal and distant from the fluconazole-resistant outbreak cluster in the same hospital. CRISPR-Cas9 editing and G. mellonella virulence assays confirmed that FKS1R658G confers echinocandin resistance in vitro and in vivo. Interestingly, the FKS1R658G mutant showed a very modest fitness cost compared with the parental WT strain, consistent with the persistence of the MDR-Cp cluster in our hospital. CONCLUSIONS: Our study showcases the emergence of MDR-Cp isolates as a novel threat in clinical settings, which undermines the efficacy of the two most widely used antifungal drugs against candidiasis, leaving only LAMB as a last resort. Additionally, surveillance studies and WGS are warranted to effectively establish infection control and antifungal stewardship strategies.

Worldwide emergence of fluconazole-resistant Candida parapsilosis: current framework and future research roadmap.

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.

Candida parapsilosis isolates carrying mutations outside FKS1 hotspot regions confer high echinocandin tolerance and facilitate the development of echinocandin resistance.

Candida parapsilosis is a significant cause of candidemia worldwide. Echinocandin-resistant (ECR) and echinocandin-tolerant (ECT) C. parapsilosis isolates have been reported in various countries but are rare. Resistance and tolerance are predominantly caused by mutations related to the hotspot (HS) regions of the FKS1 gene. A relatively high proportion of clinical C. parapsilosis isolates carrying mutations outside the HS regions has been noted in some studies, but an association with echinocandin (EC) resistance or tolerance was not explored. Herein, CRISPR-Cas9 was used and the association between amino acid substitution in FKS1 outside HS 1/2 (V595I, S745L, M1328I, F1386S, and A1422G) with EC susceptibility profile was delineated. None of the mutations conferred EC resistance, but they resulted in a significantly higher level of EC tolerance than the parental isolate, ATCC 22019. When incubated on agar plates containing ECs, specifically caspofungin and micafungin, ECR colonies were exclusively observed among ECT isolates, particularly mutants carrying V595I, S745L, and F1386S. Additionally, mutants had significantly better growth rates in yeast extract peptone dextrose (YPD) and YPD containing agents inducing membrane and oxidative stresses. The mutants had a trivial fitness cost in the Galleria mellonella model relative to ATCC 22019. Collectively, this study supports epidemiological studies to catalog mutations occurring outside the HS regions of FKS1, even if they do not confer EC resistance. These mutations are important as they potentially confer a higher level of EC tolerance and a higher propensity to develop EC resistance, therefore unveiling a novel mechanism of EC tolerance in C. parapsilosis. The identification of EC tolerance in C. parapsilosis may have direct clinical benefit in patient management.

Macrophage internalization creates a multidrug-tolerant fungal persister reservoir and facilitates the emergence of drug resistance.

Candida glabrata is a major fungal pathogen notable for causing recalcitrant infections, rapid emergence of drug-resistant strains, and its ability to survive and proliferate within macrophages. Resembling bacterial persisters, a subset of genetically drug-susceptible C. glabrata cells can survive lethal exposure to the fungicidal echinocandin drugs. Herein, we show that macrophage internalization induces cidal drug tolerance in C. glabrata, expanding the persister reservoir from which echinocandin-resistant mutants emerge. We show that this drug tolerance is associated with non-proliferation and is triggered by macrophage-induced oxidative stress, and that deletion of genes involved in reactive oxygen species detoxification significantly increases the emergence of echinocandin-resistant mutants. Finally, we show that the fungicidal drug amphotericin B can kill intracellular C. glabrata echinocandin persisters, reducing emergence of resistance. Our study supports the hypothesis that intra-macrophage C. glabrata is a reservoir of recalcitrant/drug-resistant infections, and that drug alternating strategies can be developed to eliminate this reservoir.

Determinants of fluconazole resistance and echinocandin tolerance in C. parapsilosis isolates causing a large clonal candidemia outbreak among COVID-19 patients in a Brazilian ICU.

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 TAC1L518F mutation and significantly overexpressed CDR1. Introduction of TAC1L518F 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 TAC1L518F and FKS1E1393G 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.

Candidemia Among Coronavirus Disease 2019 Patients in Turkey Admitted to Intensive Care Units: A Retrospective Multicenter Study.

Background: We evaluated the epidemiology of candidemia among coronavirus disease 2019 (COVID-19) patients admitted to intensive care units (ICUs). Methods: We conducted a retrospective multicenter study in Turkey between April and December 2020. Results: Twenty-eight of 148 enrolled patients developed candidemia, yielding an incidence of 19% and incidence rate of 14/1000 patient-days. The probability of acquiring candidemia at 10, 20, and 30 days of ICU admission was 6%, 26%, and 50%, respectively. More than 80% of patients received antibiotics, corticosteroid, and mechanical ventilation. Receipt of a carbapenem (odds ratio [OR] = 6.0, 95% confidence interval [CI] = 1.6-22.3, P = .008), central venous catheter (OR = 4.3, 95% CI = 1.3-14.2, P = .02), and bacteremia preceding candidemia (OR = 6.6, 95% CI = 2.1-20.1, P = .001) were independent risk factors for candidemia. The mortality rate did not differ between patients with and without candidemia. Age (OR = 1.05, 95% CI = 1.01-1.09, P = .02) and mechanical ventilation (OR = 61, 95% CI = 15.8-234.9, P < .0001) were independent risk factors for death. Candida albicans was the most prevalent species overall. In Izmir, Candida parapsilosis accounted for 50% (2 of 4) of candidemia. Both C parapsilosis isolates were fluconazole nonsusceptible, harbored Erg11-Y132F mutation, and were clonal based on whole-genome sequencing. The 2 infected patients resided in ICUs with ongoing outbreaks due to fluconazole-resistant C parapsilosis. Conclusions: Physicians should be aware of the elevated risk for candidemia among patients with COVID-19 who require ICU care. Prolonged ICU exposure and ICU practices rendered to COVID-19 patients are important contributing factors to candidemia. Emphasis should be placed on (1) heightened infection control in the ICU and (2) developing antibiotic stewardship strategies to reduce irrational antimicrobial therapy.

Candidemia among Hospitalized Pediatric Patients Caused by Several Clonal Lineages of Candida parapsilosis.

Candida parapsilosis is the second most common cause of candidemia in some geographical areas and in children in particular. Yet, the proportion among children varies, for example, from 10.4% in Denmark to 24.7% in Tehran, Iran. As this species is also known to cause hospital outbreaks, we explored if the relatively high number of C. parapsilosis pediatric cases in Tehran could in part be explained by undiscovered clonal outbreaks. Among 56 C. parapsilosis complex isolates, 50 C. parapsilosis were genotyped by Amplified Fragment Length Polymorphism (AFLP) fingerprinting and microsatellite typing and analyzed for nucleotide polymorphisms by FKS1 and ERG11 sequencing. AFLP fingerprinting grouped Iranian isolates in two main clusters. Microsatellite typing separated the isolates into five clonal lineages, of which four were shared with Danish isolates, and with no correlation to the AFLP patterns. ERG11 and FKS1 sequencing revealed few polymorphisms in ERG11 leading to amino-acid substitutions (D133Y, Q250K, I302T, and R398I), with no influence on azole-susceptibilities. Collectively, this study demonstrated that there were no clonal outbreaks at the Iranian pediatric ward. Although possible transmission of a diverse C. parapsilosis community within the hospital cannot be ruled out, the study also emphasizes the necessity of applying appropriately discriminatory methods for outbreak investigation.

Determinants of fluconazole resistance and the efficacy of fluconazole and milbemycin oxim combination against Candida parapsilosis clinical isolates from Brazil and Turkey.

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 TAC1L518F 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.

Clonal Candidemia Outbreak by Candida parapsilosis Carrying Y132F in Turkey: Evolution of a Persisting Challenge.

As the second leading etiological agent of candidemia in Turkey and the cause of severe fluconazole-non-susceptible (FNS) clonal outbreaks, Candida parapsilosis emerged as a major health threat at Ege University Hospital (EUH). Evaluation of microbiological and pertinent clinical profiles of candidemia patients due to C. parapsilosis in EUH in 2019-2020. Candida parapsilosis isolates were collected from blood samples and identified by sequencing internal transcribed spacer ribosomal DNA. Antifungal susceptibility testing was performed in accordance with CLSI M60 protocol and ERG11 and HS1/HS2-FKS1 were sequenced to explore the fluconazole and echinocandin resistance, respectively. Isolates were typed using a multilocus microsatellite typing assay. Relevant clinical data were obtained for patients recruited in the current study. FNS C. parapsilosis isolates were recovered from 53% of the patients admitted to EUH in 2019-2020. Y132F was the most frequent mutation in Erg11. All patients infected with C. parapsilosis isolates carrying Y132F, who received fluconazole showed therapeutic failure and significantly had a higher mortality than those infected with other FNS and susceptible isolates (50% vs. 16.1%). All isolates carrying Y132F grouped into one major cluster and mainly recovered from patients admitted to chest diseases and pediatric surgery wards. The unprecedented increase in the number of Y132F C. parapsilosis, which corresponded with increased rates of fluconazole therapeutic failure and mortality, is worrisome and highlights the urgency for strict infection control strategies, antifungal stewardship, and environmental screening in EUH.

A High Rate of Recurrent Vulvovaginal Candidiasis and Therapeutic Failure of Azole Derivatives Among Iranian Women.

Recurrent vulvovaginal candidiasis (RVVC) is one of the most prevalent fungal infections in humans, especially in developing countries; however, it is underestimated and regarded as an easy-to-treat condition. RVVC may be caused by dysbiosis of the microbiome and other host-, pathogen-, and antifungal drug-related factors. Although multiple studies on host-related factors affecting the outcome have been conducted, such studies on Candida-derived factors and their association with RVVC are lacking. Thus, fluconazole-tolerant (FLZT) isolates may cause fluconazole therapeutic failure (FTF), but this concept has not been assessed in the context of Candida-associated vaginitis. Iran is among the countries with the highest burden of RVVC; however, comprehensive studies detailing the clinical and microbiological features of this complication are scarce. Therefore, we conducted a 1-year prospective study with the aim to determine the RVVC burden among women referred to a gynecology hospital in Tehran, the association of the previous exposure to clotrimazole and fluconazole with the emergence of FLZT and fluconazole-resistant (FLZR) Candida isolates, and the relevance of these phenotypes to FTF. The results indicated that about 53% of the patients (43/81) experienced RVVC. Candida albicans and C. glabrata constituted approximately 90% of the yeast isolates (72 patients). Except for one FLZT C. tropicalis isolate, FLZR and FLZT phenotypes were detected exclusively in patients with RVVC; among them, 27.9% (12/43) harbored FLZR strains. C. albicans constituted 81.2% of FLZR (13/16) and 100% of the FLZT (13/13) isolates, respectively, and both phenotypes were likely responsible for FTF, which was also observed among patients with RVVC infected with fluconazole-susceptible isolates. Thus, FTF could be due to host-, drug-, and pathogen-related characteristics. Our study indicates that FLZT and FLZR isolates may arise following the exposure to over-the-counter (OTC) topical azole (clotrimazole) and that both phenotypes can cause FTF. Therefore, the widespread use of OTC azoles can influence fluconazole therapeutic success, highlighting the necessity of controlling the use of weak topical antifungals among Iranian women.

Epidemiology of candidemia in Shiraz, southern Iran: A prospective multicenter study (2016-2018).

Systematic candidemia studies, especially in southern Iran, are scarce. In the current prospective study, we investigated candidemia in three major healthcare centers of Shiraz, the largest city in southern Iran. Yeast isolates from blood and other sterile body fluids were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and subjected to antifungal susceptibility testing (AFST) using the broth microdilution method. Clinical data were retrieved from patients' medical records. In total, 113 yeast isolates were recovered from 109 patients, over 60% of whom received fluconazole. Antifungal drugs were prescribed without considering species identification or AFST. The all-cause mortality rate was 28%. Almost 30% of the patients were from intensive care units (ICUs). Candida albicans (56/113; 49.5%) was the most prevalent species followed by C. glabrata (26/113; 23%), C. parapsilosis (13/113; 11.5%), C. tropicalis (7/113; 6.2%), and C. dubliniensis (5/113; 4.4%). Only five isolates showed antifungal resistance or decreased susceptibility to fluconazole: one C. orthopsilosis isolate from an azole-naïve patient and two C. glabrata, one C. albicans, and one C. dubliniensis isolates from patients treated with azoles, who developed therapeutic failure against azoles later. Our results revealed a low level of antifungal resistance but a notable rate of azole therapeutic failure among patients with candidemia due to non-albicans Candida species, which threaten the efficacy of fluconazole, the most widely used antifungal in southern regions of Iran. Candidemia studies should not be confined to ICUs and treatment should be administered based on species identification and AFST results.

Landscape of candidemia is blurred in Iran, and only two studies from Tehran have extensively explored the epidemiology of candidemia. However, candidemia data from the other regions are notoriously scarce, which precludes from reaching a consensus regarding species distribution, the burden of antifungal resistance, and the clinical features of infected patients. Therefore, we conducted the current prospective candidemia study in Shiraz, one of the largest cities located in the south of Iran, from April 2016 to April 2018. More than 63% of the candidemia infections were treated by fluconazole and species identification and antifungal susceptibility testing were not used for decision making regarding the choice of antifungal treatment. Approximately 70% of the candidemia cases occurred in the wards outside of the ICUs. Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. dubliniensis were the five leading causative agents of candidemia. Antifungal resistance was rare and fluconazole resistance and/or non-wild type phenotypes were noticed in five isolates, only one was C. albicans and the rest were non-albicans Candida (NAC) species, including C. glabrata, C. dubliniensis, and C. orthopsilosis. Except for C. orthopsilosis, which was isolated from an azole-naïve patient, the rest of isolates were recovered from patients treated with azoles and all showed therapeutic failure to azoles. Collectively, our data will complete the candidemia picture in Iran and show that, although the level of resistance was rare, the therapeutic failure was notable among NAC species, which threatens the efficacy of fluconazole, the most widely used antifungal in Southern regions of Iran. Moreover, we showed that candidemia is poorly managed in Iran since species identification tools along with antifungal susceptibility testing were not used to select appropriate antifungal treatment.

SeqEditor: an application for primer design and sequence analysis with or without GTF/GFF files.

MOTIVATION: Sequence analyses oriented to investigate specific features, patterns and functions of protein and DNA/RNA sequences usually require tools based on graphic interfaces whose main characteristic is their intuitiveness and interactivity with the user's expertise, especially when curation or primer design tasks are required. However, interface-based tools usually pose certain computational limitations when managing large sequences or complex datasets, such as genome and transcriptome assemblies. Having these requirments in mind we have developed SeqEditor an interactive software tool for nucleotide and protein sequences' analysis. RESULT: SeqEditor is a cross-platform desktop application for the analysis of nucleotide and protein sequences. It is managed through a Graphical User Interface and can work either as a graphical sequence browser or as a fasta task manager for multi-fasta files. SeqEditor has been optimized for the management of large sequences, such as contigs, scaffolds or even chromosomes, and includes a GTF/GFF viewer to visualize and manage annotation files. In turn, this allows for content mining from reference genomes and transcriptomes with similar efficiency to that of command line tools. SeqEditor also incorporates a set of tools for singleplex and multiplex PCR primer design and pooling that uses a newly optimized and validated search strategy for target and species-specific primers. All these features make SeqEditor a flexible application that can be used to analyses complex sequences, design primers in PCR assays oriented for diagnosis, and/or manage, edit and personalize reference sequence datasets. AVAILABILITYAND IMPLEMENTATION: SeqEditor was developed in Java using Eclipse Rich Client Platform and is publicly available at https://gpro.biotechvana.com/download/SeqEditor as binaries for Windows, Linux and Mac OS. The user manual and tutorials are available online at https://gpro.biotechvana.com/tool/seqeditor/manual. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genetically related micafungin-resistant Candida parapsilosis blood isolates harbouring novel mutation R658G in hotspot 1 of Fks1p: a new challenge?

BACKGROUND: Echinocandin resistance rarely occurs in clinical Candida parapsilosis isolates and the underlying mechanism is unknown. OBJECTIVES: To determine the prevalence of echinocandin resistance and the underlying mechanism for a large collection of C. parapsilosis blood isolates and to determine whether the echinocandin-resistant isolates were clonally related. METHODS: C. parapsilosis blood isolates (n = 213) were subjected to antifungal susceptibility testing (CLSI M27), for micafungin, anidulafungin, amphotericin B and, if appropriate, caspofungin. Hotspot (HS) 1 and HS2 of FKS1 were sequenced for all isolates (n = 213) and microsatellite typing was performed for echinocandin-resistant isolates. RESULTS: All isolates were susceptible to amphotericin B and two isolates were intermediate to anidulafungin (MIC = 4 mg/L), while micafungin resistance was noted in four isolates (MIC >8 mg/L); three of which were also fluconazole resistant and therefore were MDR. Interestingly, micafungin-resistant isolates, but not those intermediate to anidulafungin, carried novel mutation R658G in HS1 of Fks1p; three of which also harboured Y132F+K143R in Erg11. The first isolate (MICR1) was recovered in November 2017 from a patient admitted to paediatric gastroenterology who showed therapeutic failure under caspofungin treatment. MICR2-MICR4 were collected during 2018-19 and were recovered from three echinocandin-naive paediatric-surgery patients; the isolates shared the same genotype. CONCLUSIONS: Herein, for the first time (to the best of our knowledge), we identified micafungin-resistant C. parapsilosis blood isolates harbouring a novel mutation in HS1 of FKS1, which was likely attributable to in vitro micafungin resistance and in vivo caspofungin therapeutic failure. The acquisition of micafungin-resistant C. parapsilosis isolates in echinocandin-naive patients likely implicates clonal expansion, as supported by the close genetic relatedness of MICR2-MICR4.

Clinical and microbiological features of candiduria in critically ill adult patients in Shiraz, Iran (2016-2018): deviations from international guidelines and fluconazole therapeutic failure.

Candiduria is common among patients admitted to intensive care units (ICUs); however, clinical and microbiological data are limited, which accounts for non-compliance with international guidelines, including over treatment of asymptomatic candiduria that promotes antifungal resistance. This prospective study included adult patients admitted to ICUs of five referral hospitals in Shiraz, Iran, during 2016-2018. Species were identified by MALDI-TOF MS, and antifungal susceptibility was assessed according to CLSI M27-A3/S4. Among 2086 patients, 162 and 293 developed candiduria and bacteriuria, respectively. In total, 174 yeast isolates were collected; 88.5% were Candida albicans (91/174; 52.2%), C. glabrata (38/174; 21.8%), and C. tropicalis (25/174; 14.3%). Antifungal resistance was rare; only two isolates (one C. tropicalis and one C. krusei) were fluconazole resistant. Symptomatic candiduria was noted in 31.4% of patients (51/162); only 37% (19/51) of them were treated and 36.82% (7/19) showed fluconazole therapeutic failure. Two symptomatic patients developed candidemia shortly after candiduria. Among asymptomatic patients, 31.5% (35/111) were overtreated with fluconazole. The mortality rate was 25.3% (41/162); it did not differ between symptomatic and asymptomatic patients. Our results indicate that deviation from standard-of-care treatment for candiduria is a matter of concern given the high rate of fluconazole therapeutic failure among patients with symptomatic candiduria. LAY SUMMARY: Candiduria is an underestimated clinical presentation among critically ill patients and detailed data are scarce in this regard. Given the high rate of fluconazole therapeutic failure and development of candidemia in some cases, the mistreatment of candiduria should not be overlooked by clinicians.

Recent Increase in the Prevalence of Fluconazole-Non-susceptible Candida tropicalis Blood Isolates in Turkey: Clinical Implication of Azole-Non-susceptible and Fluconazole Tolerant Phenotypes and Genotyping.

Candida tropicalis is the fourth leading cause of candidemia in Turkey. Although C. tropicalis isolates from 1997 to 2017 were characterized as fully susceptible to antifungals, the increasing global prevalence of azole-non-susceptible (ANS) C. tropicalis and the association between high fluconazole tolerance (HFT) and fluconazole therapeutic failure (FTF) prompted us to re-evaluate azole susceptibility of C. tropicalis in Turkey. In this study, 161 C. tropicalis blood isolates from seven clinical centers were identified by ITS rDNA sequencing, genotyped by multilocus microsatellite typing, and tested for susceptibility to five azoles, two echinocandins, and amphotericin B (AMB); antifungal resistance mechanisms were assessed by sequencing of ERG11 and FKS1 genes. The results indicated that C. tropicalis isolates, which belonged to 125 genotypes grouped into 11 clusters, were fully susceptible to echinocandins and AMB; however, 18.6% of them had the ANS phenotype but only two carried the ANS-conferring mutation (Y132F). HFT was recorded in 52 isolates, 10 of which were also ANS. Large proportions of patients infected with ANS and HFT isolates (89 and 40.7%, respectively) showed FTF. Patients infected with azole-susceptible or ANS isolates did not differ in mortality, which, however, was significantly lower for those infected with HFT isolates (P = 0.007). There were significant differences in mortality (P = 0.02), ANS (P = 0.012), and HFT (P = 0.007) among genotype clusters. The alarming increase in the prevalence of C. tropicalis blood isolates with ANS and HFT in Turkey and the notable FTF rate should be a matter of public health concern.

The Quiet and Underappreciated Rise of Drug-Resistant Invasive Fungal Pathogens.

Human fungal pathogens are attributable to a significant economic burden and mortality worldwide. Antifungal treatments, although limited in number, play a pivotal role in decreasing mortality and morbidities posed by invasive fungal infections (IFIs). However, the recent emergence of multidrug-resistant Candida auris and Candida glabrata and acquiring invasive infections due to azole-resistant C. parapsilosis, C. tropicalis, and Aspergillus spp. in azole-naïve patients pose a serious health threat considering the limited number of systemic antifungals available to treat IFIs. Although advancing for major fungal pathogens, the understanding of fungal attributes contributing to antifungal resistance is just emerging for several clinically important MDR fungal pathogens. Further complicating the matter are the distinct differences in antifungal resistance mechanisms among various fungal species in which one or more mechanisms may contribute to the resistance phenotype. In this review, we attempt to summarize the burden of antifungal resistance for selected non-albicansCandida and clinically important Aspergillus species together with their phylogenetic placement on the tree of life. Moreover, we highlight the different molecular mechanisms between antifungal tolerance and resistance, and comprehensively discuss the molecular mechanisms of antifungal resistance in a species level.