Antifungal Resistance Research Articles

Yeast-contaminated water as a potential emerging health concern: a review

Invasive fungal infections pose a serious risk to human health; therefore, it is important to study the dissemination and proliferation of pathogenic fungal species in the environment. This could prove useful in preventing infections in susceptible individuals, such as those who are immune-compromised or suppressed. Pathogenic yeasts belonging to the genera Candida, Cryptococcus and Rhodotorula are commonly found in various water sources that are used for daily activities and are included in the World Health Organization fungal priority pathogens list, further warranting investigation into the possibility that infections may occur through contact with yeast-contaminated water. In addition, the close association between antifungal pollutants and yeast in water may induce acquired antifungal resistance development, further complicating the effective treatment of these infections. Thus, investigating the presence and antifungal susceptibility of yeast found in water and identifying ways to monitor potential fungal pathogens may prove useful in combating invasive fungal infections. This review deals with the occurrence and infection risks posed by pathogenic yeasts in water as well as the possibility of these yeasts acquiring antifungal resistance due to the simultaneous presence of antifungal compounds from medical and agricultural runoff.

The Candida auris Infection After the COVID-19 Pandemic Seems to be an Urgent Public Health Emergency: A Call to Attention.

Inconsequential diseases can sometimes become extremely dangerous through mutation. Antifungal resistance has increased by 24%, and resistance only due to Candida auris (C. auris) species have increased by 60%. Here, we aimed to assess the knowledge of antifungal treatment and preventative measures to mitigate the consequences of infections caused by C. auris. We performed a comprehensive literature search and gathered information for this review from publicly available published articles. We used C. auris, C. auris infection, mycoses, and antifungal resistance as search terms in Google Scholar, PubMed, and Scopus. We extracted the relevant information from the available article after careful evaluation. The genus C. auris was especially difficult to diagnose, as it was often mistaken for other types of yeast and led to incorrect treatment. The only effective method for diagnosing C. auris is through blood culture testing in laboratories and PCR tests. Because of its mutation, C. auris has grown increasingly resistant to all three classes of antifungals, with almost all strains resistant to fluconazole. These resistances were traced back to multiple-point mutations in certain genes, such as ERG11 in the case of fluconazole and amphotericin B. In addition, C. auris is phylogenetically related to C. haemulonii, an emerging pathogen notably resistant to antifungals. So, it may be an evolutionary resistance that occurred earlier but has only begun to spread now. Echinoderm, a reliable treatment for C. auris infections showed ineffectively against FKS1 and ERG3 gene mutated Candida strains. Newly developed antifungal agents, like Ibrexafungerp, showed promising results against echinocandin-resistant strains in clinical trials. Without vaccines and effective treatment, its capacity to mutate and spread has detrimental effects on humans. Therefore, extensive research on drug development, quick, reliable diagnosis, and effective strategies for disease prevention and treatment are required to mitigate the health impact of C. auris.

Improved Broad Spectrum Antifungal Drug Synergies with Cryptomycin, a Cdc50-Inspired Antifungal Peptide.

Fungal infections in humans are difficult to treat, with very limited drug options. Due to a confluence of factors, there is an urgent need for innovation in the antifungal drug space, particularly to combat increasing antifungal drug resistance. Our previous studies showed that Cdc50, a subunit of fungal lipid translocase (flippase), is essential for Cryptococcus neoformans virulence and required for antifungal drug resistance, suggesting that fungal lipid flippase could be a novel drug target. Here, we characterized an antifungal peptide, Cryptomycinamide (KKOO-NH2), derived from a 9-amino acid segment of the C. neoformans Cdc50 protein. A fungal killing assay indicated that KKOO-NH2 is fungicidal against C. neoformans. The peptide has antifungal activity against multiple major fungal pathogens with a minimum inhibitory concentration (MIC) of 8 μg/mL against C. neoformans and Candida glabrata, 16 μg/mL against Candida albicans and C. auris, and 32 μg/mL against Aspergillus fumigatus. The peptide has low cytotoxicity against host cells based on our hemolysis assays and vesicle leakage assays. Strikingly, the peptide exhibits strong drug synergy with multiple antifungal drugs, including amphotericin B, itraconazole, and caspofungin, depending on the specific species on which the combinations were assayed. The fluorescently labeled peptide was detected to localize to the plasma membrane, likely inhibiting key interactions of Cdc50 with membrane proteins such as P4 ATPases. Cryptococcus cells exposed to sub-MIC of peptide showed increased reactive oxygen species production and intracellular calcium levels, indicating a peptide-induced stress response. Decreased intracellular proliferation within macrophages was observed after 30 min of peptide exposure and 24 h coincubation with macrophages, providing a potential translational mechanism to explore further in vivo. In aggregate, the synergistic activity of our KKOO-NH2 peptide may offer a potential novel candidate for combination therapy with existing antifungal drugs.

Collateral sensitivity counteracts the evolution of antifungal drug resistance in Candida auris.

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.

Application of Raman spectroscopy and machine learning for Candida auris identification and characterization.

Currently, combating Candida auris infections and transmission is challenging due to the lack of efficient identification and characterization methods for this species. To address these challenges, our study presents a novel approach that utilizes Raman spectroscopy and artificial intelligence to achieve precise identification and characterization of C. auris at the singe-cell level. It can accurately identify a single cell from the four C. auris clades. Additionally, we developed machine learning models designed to detect antifungal resistance in C. auris cells and differentiate between two distinct phenotypes based on the single-cell Raman spectrum. We also constructed prediction models for detecting aggregating and filamentous cells in C. auris, both of which are closely linked to its virulence. These results underscore the merits of Raman spectroscopy in the identification and characterization of C. auris, promising improved outcomes in the battle against C. auris infections and transmission.

Characterization of Yeast Isolated from the Gut Microbiota of Tunisian Children with Autism Spectrum Disorder

Research on the microbiota–gut–brain axis in autism has primarily focused on bacteria, with limited attention to fungi. There is a growing interest in understanding the involvement of fungi, particularly Candida, in patients with autism spectrum disorder. The aim of this study was to assess the prevalence, antifungal susceptibility profiles and virulence factors of Candida isolates from the guts of Tunisian children with autism. Twenty-eight children with autism and forty-six controls were enrolled. Candida isolates from the faecal samples were identified using biochemical and molecular methods; antifungal susceptibility testing was determined by the EUCAST broth microdilution method and virulence factors, including biofilm formation, cell surface hydrophobicity and phospholipase and proteinase activities, were assessed in vitro. As a result, Candida was detected in 13 children with autism (46.4%) and 14 control children (30.4%). Candida albicans was found to be the most common species isolate in the faeces of both groups of children. Antifungal susceptibility profiles showed that one Candida isolate was resistant to amphotericin B and anidulafungin (3.7%), six were resistant to micafungin (22.2%) and five were resistant to fluconazole (18.5%). All Candida isolates were biofilm producers. Of the twenty-seven isolates, only four showed phospholipase activity (14.8%), eight showed aspartyl-proteinase activity (29.6%) and nine were hydrophobic (33.3%). These results highlight the presence of Candida in the guts of children with autism, as well as the ability to express multiple virulence factors and the antifungal resistance, and they emphasize the need for further studies to confirm intestinal Candida colonization and its potential role in autism.

Candida albicans isolates contain frequent heterozygous structural variants and transposable elements within genes and centromeres.

The human fungal pathogen Candida albicans poses a significant burden on global health, causing high rates of mortality and antifungal drug resistance. C. albicans is a heterozygous diploid organism that reproduces asexually. Structural variants (SVs) are an important source of genomic rearrangement, particularly in species that lack sexual recombination. To comprehensively investigate SVs across clinical isolates of C. albicans, we conducted long read sequencing and genome-wide SV analysis in three distantly related clinical isolates. Our work included a new, comprehensive analysis of transposable element (TE) composition, location and diversity. SVs and TEs are frequently close to coding sequences and many SVs are heterozygous, suggesting that SVs might impact gene and allele-specific expression. Most SVs are uniquely present in only one clinical isolate, indicating that SVs represent a significant source of intra-species genetic variation. We identified multiple, distinct SVs at the centromeres of Chromosome 4 and Chromosome 5, including inversions and transposon polymorphisms. These two chromosomes are often aneuploid in drug resistant clinical isolates, and can form isochromosome structures with breakpoints near the centromere. Further screening of 100 clinical isolates confirmed the widespread presence of centromeric SVs in C. albicans, often appearing in a heterozygous state, indicating that SVs are contributing to centromere evolution in C. albicans Together, these findings highlight that SVs and TEs are common across diverse clinical isolates of C. albicans and that the centromeres of this organism are important sites of genome rearrangement.

CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans.

Candida albicans, an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (Guide RNAs with Integrated Repair Templates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in C. albicans. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.

Investigating role of positively selected genes and mutation sites of ERG11 in drug resistance of Candida albicans.

The steep increase in acquired drug resistance in Candida isolates has posed a great challenge in the clinical management of candidiasis globally. Information of genes and codon sites that are positively selected during evolution can provide insights into the mechanisms driving antifungal resistance in Candida. This study aimed to create a manually curated list of genes of Candida spp. reported to be associated with antifungal resistance in literature, and further investigate the structure-function implications of positively selected genes and mutation sites. Sequence analysis of antifungal drug resistance associated gene sequences from various species and strains of Candida revealed that ERG11 and MRR1 of C. albicans were positively selected during evolution. Four sites in ERG11 and two sites in MRR1 of C. albicans were positively selected and associated with drug resistance. These four sites (132, 405, 450, and 464) of ERG11 are predictive markers for azole resistance and have evolved over time. A well-characterized crystal structure of sterol-14-α-demethylase (CYP51) encoded by ERG11 is available in PDB. Therefore, the stability of CYP51 in complex with fluconazole was evaluated using MD simulations and molecular docking studies for two mutations (Y132F and Y132H) reported to be associated with azole resistance in literature. These mutations induced high flexibility in functional motifs of CYP51. It was also observed that residues such as I304, G308, and I379 of CYP51 play a critical role in fluconazole binding affinity. The insights gained from this study can further guide drug design strategies addressing antimicrobial resistance.

RNAi epimutations conferring antifungal drug resistance are inheritable.

Epimutations modify gene expression and lead to phenotypic variation while the encoding DNA sequence remains unchanged. Epimutations mediated by RNA interference (RNAi) and/or chromatin modifications can confer antifungal drug resistance and may impact virulence traits in fungi (1-5). However, whether these epigenetic modifications can be transmitted across generations following sexual reproduction was unclear. This study demonstrates that RNAi epimutations conferring antifungal drug resistance are transgenerationally inherited in the human fungal pathogen Mucor circinelloides . Our research revealed that RNAi-based antifungal resistance follows a DNA sequence-independent, non-Mendelian inheritance pattern. Small RNAs (sRNAs) are the exclusive determinants of inheritance, transmitting drug resistance independently of other known repressive epigenetic modifications. Unique sRNA signature patterns can be traced through inheritance from parent to progeny, further supporting RNA as an alternative molecule for transmitting information across generations. This study marks a significant advance in understanding epigenetic inheritance, highlighting RNAi-exclusive epimutations as a widespread phenomenon in the pathogenic M. circinelloides species complex. Understanding how epimutations occur, propagate, and confer resistance may enable their detection in other eukaryotic pathogens, provide solutions for challenges posed by rising antimicrobial drug resistance (AMR), and also advance research on phenotypic adaptability and its evolutionary implications.

Do morphogenetic switching and intraspecies variation enhance virulence of Candida auris?

Intraspecies variations that affect pathogenicity and antifungal resistance traits pose a serious obstacle to efficient therapy of Candida auris infections. Recent reports indicate that mutations determine drug susceptibility and virulence. However, mutations alone cannot fully explain a bewildering variety of phenotypes in clinical isolates from known C. auris clades, suggesting an unprecedented complexity underlying virulence traits and antifungal resistance. Hence, we wish to discuss how phenotypic plasticity promotes morphogenetic switching and how that contributes to intraspecies variations in the human fungal pathogen C. auris. Further, we will also discuss how intraspecies variations and morphogenetic events can impact the progress in molecular mycology research that aims to find better treatments for C. auris infections. Finally, we will present our opinion as to the most relevant questions to be addressed when trying to better understand the pathophysiology of C. auris.

Antifungal Potential of Struchium sparganophora (Antsbush): A Promising Candidate for Treating Candidiasis and Cryptococcosis

Aims: The incidence of Candidiasis and Cryptococcosis, and the emergence of acquired antifungal resistance have increased rapidly. This study aimed to evaluate the effectiveness of S. sparganophora leaf extracts against C. albicans and C. neoformans; and to determine whether there was any difference between the antifungal effects of S. sparganophora leaf extracts and two conventional antifungal agents (AFAs). Study Design: Experiment-based study. Place and Duration of Study: S. sparganophora leaves was obtained from a site in Kimbia, Berbice River, Guyana; identified by the Biodiversity Centre and tested at the Main Laboratory at the College of Medical Sciences, University of Guyana during January- August 2023. Methodology: Dried pulverised leaves were macerated using different solvents and concentrated using a rotary evaporator. Sterile filter paper discs were soaked in different concentrations of the various extracts. Antifungal discs were placed on Sabouraud’s Dextrose Agar plates seeded with fungi. All plates were incubated and zones of inhibitions (ZOIs) were measured and expressed as mean ± SD. Results: S. sparganophora leaf extracts showed large ZOI especially at the 100 mg/ml concentrations against all organisms tested. The largest ZOI was seen for the hexane extract against C. neoformans (35.5±5 mm) and C. Albicans In-House B (18.5±3.5 mm) at 100 mg/ml concentrations. No ZOI was observed against the AFA fluconazole by C. albicans In-House B, while large ZOI were observed with the hexane and ethyl acetate extracts at 100 mg/ml concentrations. Statistical analysis showed correlation between the ZOI and concentrations, ZOI and solvent extract, and ZOI and type of fungi. Conclusion: In conclusion, S. sparganophora leaf extracts have great antifungal activity and in some cases showed greater activity when compared to conventional AFAs.

Molecular and Microbiological Characterization of Candida auris Strains Isolated from Colonized and Infected Patients in a New York City Tertiary Care Center

Abstract Candida auris is a multidrug resistant pathogen that causes healthcare associated invasive infections and outbreaks with high mortality rates, often involving intensive care units (ICU). High rates of C. auris colonization among patients in ICUs are associated with increased risk of subsequent C. auris hospital-acquired infections. In this study, microbiological characteristics, and antifungal susceptibility patterns of C. auris isolates collected between January and December 2023 were investigated. Whole-genome sequencing was used to examine the genetic variation and molecular epidemiological characteristics. During the study period, a total of 20 patients were found to be colonized with C. auris by three site skin swab and eight of these patients subsequently developed invasive infection with C. auris. Among patients with both colonization and infection, all invasive isolates were closely related genetically to colonizing isolates and belonged to clade I. Candidemia was the most common infection identified in 4/8 (50%) patients. All isolates were resistant to fluconazole and thirteen isolates from six patients were resistant to amphotericin B. Paired isolates from patients with both colonization and invasive infection showed the same antifungal resistance patterns. All isolates had the ERG11 p.K143R mutation known to cause fluconazole resistance. Three isolates from one patient with a FKS1 hotspot region 3 p.M690I mutation were resistant to caspofungin and two isolates from one patient with a FKS1 hotspot region 2 p.R1354H mutation were resistant to caspofungin, anidulafungin and micafungin. Our study demonstrates that colonization with C. auris is associated with increased risk of infection by the same strain amongst critical ill patients. C. auris screening and implementation of adequate infection control measures are urgently needed to prevent the dissemination of C. auris infections.

Antifungal Resistance and Breakthrough Candidemia Among Pediatric Cancer Patients: An Emerging Threat

Abstract Background Cancer patients are at risk for candidemia. Resistance and breakthrough Candida represent an emerging threat. The study aims to analyze the clinical features, resistance patterns and breakthrough candidemia among pediatric cancer patients. Methods The retrospective study was conducted from 2014 - 2020 in children admitted to Children Cancer Hospital, Egypt. Clinical, microbiological, and outcomes were analyzed. Mortality was estimated within 30 days from Candida spp. Isolation from blood. Results Three hundred fifty (350) pediatric cancer patients were diagnosed with candidemia. The median age was four years (4 months- 18 years). Most of the patients had hematological malignancies (54%) while solid tumors represent (46%). Clinical features of the patients included neutropenia (80%), the presence of central venous line (57%), ICU admission (44%), and steroid use (43%). Non-Albicans candida was predominant (71%) compared to Candida Albicans (29%). The isolated species were: C. Albicans (29%), C.Tropicalis (29%), C. parapsilosis (21%), C. krusei (7%), C.glabrata(3%), C. guilliermondii (1.5%)and C. famata (2%). Chronic disseminated candidiasis was reported in 30 (9%) patients, with the liver being the most affected organ. Breakthrough candidemia on top of echinocandin prophylaxis was reported among 62(18%) patients with C. parapsilosis is the most common 31(50%) isolated pathogen. From 96 patients with breakthrough candidemia, all-cause mortality was reported in 32 patient (33%) while candida attributable cause of death in elven (11%) patients. Antifungal resistance was reported in 74(21%) patients: fluconazole (15%), liposomal ampho-B (6%), echinocandin (5%) and voriconazole (3%). Among 74 patients with resistant candidemia, death was reported in 24%. Echinocandin Resistant candida was a direct cause of mortality in 3(17%) patients. The overall day- 30 mortality rate was 24% (85/350 patients), while candida attributable of mortality was (10%). Patients with septic shock, ICU admission, and resistant candida had a higher mortality risk. Conclusion Echinocandin-resistant and breakthrough Candida are significant threat with a high mortality rate among pediatric cancer patients. Antifungal stewardship may be helpful. Figure 1. Resistance Pattern of Candidemia among Pediatric Cancer Patients

Prevalence, Species Distribution and Resistance of Candidemia in Pediatric and Adult Patients in a Northeast Italy University Hospital.

Candidemia and invasive candidiasis (IC) are causes of morbidity and mortality in healthcare settings, with notable differences between children and adults. Understanding the species distribution and antimicrobial susceptibility profiles of clinical isolates can guide empiric therapy in patients at risk of IC. This study investigated the incidence and antifungal susceptibility patterns of yeasts involved in IC in pediatric and adult patients from 2019 to 2023. The average incidence of IC was 0.715 per 1000 patients, increasing over the study period; infants had the highest incidence rates. Over half of the IC episodes occurred in intensive care units (ICUs). Non-albicans Candida (NAC) species represented the most frequently isolated species in adults and children (55.96% and 50.0%, respectively), with the prevalence of C. parapsilosis (26.45% and 14.7%, respectively), N. glabratus (14.97% and 8.82%, respectively) and C. tropicalis (4.36% and 2.94%, respectively). C. lusitaniae was identified in 14.7% of pediatric IC cases. In NAC species, antifungal resistance has also increased over the five years of the study: 69.12% were resistant to azoles and 7.35% were resistant to micafungin. Resistance was higher in pediatric patients. Our study highlights differences in IC characteristics between pediatric and adult populations and emphasizes the importance of targeted antifungal stewardship in ICU patients with NAC invasive infections.

Chemical Survey and Antifungal Efficacy of Sargassum muticum's Alkaloids and Phenolic-Rich Fraction Against Airborne Toxigenic and Nosocomial Opportunistic Molds Isolates.

The Atlantic coastline of El-Jadida, Morocco, is renowned for its plentiful algae, especially brown seaweed, which is rich in active compounds known for their antifungal properties. This valuable resource offers an exciting opportunity to tackle the numerous challenges posed by invasive fungal infections, allergies, mycotoxin-related food poisoning, and drug-resistant strains. Underscoring the urgent need to explore alternative, sustainable, and environmentally friendly antifungal agents derived from algae. This study aimed to evaluate the antifungal activity of total alkaloids and phenolic-rich fractions derived from seven species of Pheophyceae: Sargassum muticum, Sargassum vulgare, Bifurcaria bifurcata, Cystoseira tamariscifolia, Cystoseira humilis, Laminaria ochroleuca, and Fucus spiralis against four fungi: airborne toxigenic isolates of Aspergillus westerdijkiae and Chaetomium globosum as well as nosocomial opportunistic isolates of Aspergillus nidulans and Scopulariopsis brevicaulis. The study also aimed to identify the most effective alga and its specific active compounds through LC-MS and GC-MS analysis. The invasive Sargassum muticum was chosen as the most potent alga in inhibiting the growth of mycelium. For the first time, the alkaloids palmatine and jatrorrhizine, along with caulerpin, have been identified. The chloroform fraction revealed the prevalence of phenolic compounds including, phenolic acids, flavonoids, and phlorotannins. The lowest minimum inhibitory concentrations (MICs), with a maximum fungal load of 108 colony-forming unit (CFU), recorded ranged from 3.12 to 6.25μg/mL by the phenolic-rich fraction against airborne toxigenic isolates, and from 100 to 200μg/mL against nosocomial opportunistic isolates by the total alkaloids. In comparison, the positive control, ketoconazole, showed higher MICs and resistance against A. nidulans. The valorization of Sargassum muticum is proposed as a green strategy to preserve the ecological balance, combat antifungal resistance, and address public health challenges.

Multi-Locus Sequence Typing-Based Genetic Analysis, Antifungal Resistance, and Clinical Prognosis of Cryptococcus neoformans Infections in HIV-Infected Patients in Northern China.

This study aimed to investigate the molecular epidemiological characteristics and drug sensitivity of Cryptococcus from HIV-infected patients and their relationship with patients' prognosis. Seventy-six strains were collected and identified to the species level by matrix-assisted laser desorption ionization-time of flight mass spectrometry, confirmed by internal transcribed spacer sequencing. Multi-locus sequence typing was used for the typing of Cryptococcus, and its antifungal susceptibility was tested using FUNGUS 3. The clinical outcomes of the patients were reviewed at 3-, 6-, 9-, and 12-month follow-ups. All strains were Cryptococcus neoformans var. grubii classified into seven sequence types (STs) dominated by ST5, ST31, and a new ST702 strain. The 6- and 9-month survival rates were highest for patients infected with ST31, ST32, and ST174. The antifungal resistant rates were 13.2%, 2.6%, and 1.4% for fluconazole, amphotericin B, and 5-fluorocytosine. Except itraconazole, the minimum inhibitory concentration (MIC) values and wild type (WT)/non-wild type (NWT) of Cryptococcus for antifungal drugs were not related to the clinical prognosis of HIV-infected patients with cryptococcal infection. ST5 was the main ST type, and the new ST702 type was found in a patient who died in a short period of time. Cryptococcus neoformans var. grubii had a relatively high antifungal drug resistance rate to fluconazole. The WT strain accounted for the highest proportions for 5-fluorocytosine, amphotericin B, fluconazole, voriconazole, and itraconazole. The MIC values of Cryptococcus for first-line antifungal drugs showed no relationship with clinical prognosis, implying that MIC values cannot be used to predict the clinical outcome of these patients.

Azole Combinations and Multi-Targeting Drugs That Synergistically Inhibit Candidozyma auris.

Limited antifungal treatment options and drug resistance require innovative approaches to effectively combat fungal infections. Combination therapy is a promising strategy that addresses these pressing issues by concurrently targeting multiple cellular sites. The drug targets usually selected for combination therapy are from different cellular pathways with the goals of increasing treatment options and reducing development of resistance. However, some circumstances can prevent the implementation of combination therapy in clinical practice. These could include the increased risk of adverse effects, drug interactions, and even the promotion of drug resistance. Furthermore, robust clinical evidence supporting the superiority of combination therapy over monotherapy is limited and underscores the need for further research. Despite these challenges, synergies detected with different antifungal classes, such as the azoles and echinocandins, suggest that treatment strategies can be optimized by better understanding the underlying mechanisms. This review provides an overview of multi-targeting combination strategies with a primary focus on Candidozyma auris infections.

Antifungal susceptibility, molecular epidemiology, and clinical risk factors of Candida glabrata in intensive care unit in a Chinese Tertiary Hospital.

The increasing incidence and high mortality rate of Candida glabrata infection in ICU patients is an important issue. Therefore, it is imperative to investigate the antifungal susceptibility profiles and epidemiological characteristics in local regions. Herein, antifungal susceptibility testing was conducted to determine the minimum inhibitory concentrations (MICs) of eight antifungal drugs. Multilocus sequence typing (MLST) was used to study the strain genotype, geographical distribution, and susceptibility to antifungal agents among C. glabrata isolates. The mechanism of echinocandin resistance was explored by sequencing the FKS1 and FKS2 genes (encoding 1,3-β-D-glucan synthases) of echinocandin-resistant C. glabrata strains. Moreover, we further investigated the clinical manifestations and the various risk factors of patients infected with C. glabrata in the ICU. We selected 234 C. glabrata isolates from 234 patients in the ICU randomly for the follow-up study. Cross-resistance was found among the ICU C. glabrata isolates. Analysis using MLST showed that the genetic diversity among the C. glabrata isolates was low. Furthermore, sequence type showed no correlation with the antifungal resistance profiles, but was associated with geographical distribution. We also revealed novel mutations in FKS1 (S629P) and FKS2 (W1497stop) that mediated high-level echinocandin resistance (MIC >8 µg/mL). More than 14 days' stay in ICU (P=0.007), Acute Physiology and Chronic Health Evaluation II (APACHE-II) score (P=0.024), prior antifungal exposure (P=0.039) and lung disease (P=0.036) were significantly associated with antifungal resistant/non-wild-type C. glabrata infection. Our study shed light on the antifungal susceptibility, molecular epidemiology, and clinical risk factors of C. glabrata in the ICU of a Chinese Tertiary Hospital. Importantly, we revealed the molecular mechanism of echinocandin resistance. These results highlight the significance of continued surveillance in ICUs and provide data support for the treatment of C. glabrata in clinics.

Characterization of Candida species isolated from clinical specimens: insights into virulence traits, antifungal resistance and molecular profiles

BackgroundCandida 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.AimThis 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.MethodA 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.ResultsThe 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.ConclusionThe 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.