Search Results (48)

The feline panleukopenia virus (FPV) is a highly contagious virus that affects cats worldwide, characterized by leukopenia, high temperature and diarrhea. Recently, the continuous prevalence and variation of FPV have attracted widespread concern. The aim of this study was to investigate the isolation, genetic evolution, molecular characterization and epidemiological analysis of FPV strains among cats and dogs in China from 2019 to 2024. The 41 FPV strains, including 38 feline strains and 3 canine strains, were isolated from rectal swab samples by inoculating monolayer FK81 cells and performing a plaque purification assay. The viral and hemagglutination titers of these 41 FPV strains were 10^4.33~10^6.33 TCID₅₀/0.1 mL and 7.0 log₂~9.7 log₂, respectively. Based on the complete VP2 gene, the nucleotide homology of these FPV strains was 98.91~100%, and the homology with 24 reference FPV strains from different countries and hosts was 98.85~100%. The phylogenetic analysis revealed that 41 FPV strains were more closely related to the FPV strains of Asian origin (Asian FPV strain group) than those of European and American origin (European and American FPV strain group). Furthermore, 12 mutation sites of the VP2 protein were found in these FPV strains, of which 91 and 232 amino acid sites were previously reported. Moreover, the 91 amino acid site was found to be a positive selection site with the highest dN/dS value in the selection pressure analysis. Importantly, 35 FPV strains with 91S substitution in the VP2 protein (FPV-VP2-91S strains) had formed obvious evolutionary branches in the Asian FPV strain group. The analysis of all available VP2 protein sequences of Chinese FPV strains in the GenBank database showed that the occurrence rate of FPV-VP2-91S strains had been increasing from 15.63% to 100% during 2017~2024, indicating that the FPV-VP2-91S substitution in the VP2 protein was a noteworthy molecular characteristic of the dominant FPV strains in China. These results contribute to a better understanding of their genetic evolution and renew the knowledge of FPV molecular epidemiology. Full article

Invasive candidiasis poses a worldwide threat because of the rising prevalence of antifungal resistance, resulting in higher rates of morbidity and mortality. Additionally, Candida species, which are opportunistic infections, have significant medical and economic consequences for immunocompromised individuals. This study explores the antifungal potential of chitosan to mitigate caspofungin resistance in caspofungin-resistant Candida albicans, C. krusei, and C. tropicalis isolates originating from human and animal sources using agar well diffusion, broth microdilution tests, and transmission electron microscope (TEM) analysis of treated Candida cells. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was performed to assess the expression of SAGA complex genes (GCN5 and ADA2) and the caspofungin resistance gene (FKS) in Candida species isolates after chitosan treatment. The highest resistance rate was observed to ketoconazole (80%) followed by clotrimazole (62.7%), fluconazole (60%), terbinafine (58%), itraconazole (57%), miconazole (54.2%), amphotericin B (51.4%), voriconazole (34.28%), and caspofungin (25.7%). Nine unique FKS mutations were detected, including S645P (n = 3 isolates), S645F, L644F, S645Y, L688M, E663G, and F641S (one isolate in each). The caspofungin minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values before chitosan treatment ranged from 2 to 8 µg/mL and 4 to 16 µg/mL, respectively. However, the MIC and MFC values were decreased after chitosan treatment (0.0625–1 µg/mL) and (0.125–2 µg/mL), respectively. Caspofungin MIC was significantly decreased (p = 0.0007) threefold following chitosan treatment compared with the MIC values before treatment. TEM analysis revealed that 0.5% chitosan disrupted the integrity of the cell surface, causing irregular morphologies and obvious aberrant changes in cell wall thickness in caspofungin-resistant and sensitive Candida isolates. The cell wall thickness of untreated isolates was 0.145 μm in caspofungin-resistant isolate and 0.125 μm in sensitive isolate, while it was significantly lower in chitosan-treated isolates, ranging from 0.05 to 0.08 μm when compared with the cell wall thickness of sensitive isolate (0.03 to 0.06 μm). Moreover, RT-qPCR demonstrated a significant (p < 0.05) decrease in the expression levels of histone acetyltransferase genes (GCN5 and ADA2) and FKS gene of caspofungin-resistant Candida species isolates treated with 0.5% chitosan when compared with before treatment (fold change values ranged from 0.001 to 0.0473 for GCN5, 1.028 to 4.856 for ADA2, and 2.713 to 12.38 for FKS gene). A comparison of the expression levels of cell wall-related genes (ADA2 and GCN5) between caspofungin-resistant and -sensitive isolates demonstrated a significant decrease following chitosan treatment (p < 0.001). The antifungal potential of chitosan enhances the efficacy of caspofungin against various caspofungin-resistant Candida species isolates and prevents the development of further antifungal resistance. The results of this study contribute to the progress in repurposing caspofungin and inform a development strategy to enhance its efficacy, appropriate antifungal activity against Candida species, and mitigate resistance. Consequently, chitosan could be used in combination with caspofungin for the treatment of candidiasis. Full article

The efficacy of different echinocandins is assessed by evaluating the in vitro activity of a novel antifungal, rezafungin, against invasive fungal isolates in comparison with anidulafungin and caspofungin. Using the broth microdilution (BMD) method, the susceptibility of 1000 clinical Candida isolates (including 400 C. albicans, 200 C. glabrata, 200 C. parapsilosis, 150 C. tropicalis and 50 C. krusei) and 150 Aspergillus isolates (100 A. fumigatus and 50 A. flavus) from the Eastern China Invasive Fungi Infection Group (ECIFIG) was tested for the antifungals including anidulafungin, rezafungin, caspofungin and fluconazole. The echinocandins showed strong activity against C. albicans that was maintained against fluconazole-resistant isolates. The GM MIC (geometric mean minimum inhibitory concentration) value of rezafungin was found to be comparable to that of anidulafungin or caspofungin against the five tested common Candida species. C. tropicalis exhibited higher resistance rates (about 8.67–40.67% in different antifungals) than the other four Candida species. Through the sequencing of FKS genes, we searched for mutations in echinocandin-resistant C. tropicalis isolates and found that all displayed alterations in FKS1 S654P. The determined MEC (minimal effective concentration) values against A. fumigatus and A. flavus for rezafungin (0.116 μg/mL, 0.110 μg/mL) are comparable to those of caspofungin (0.122 μg/mL, 0.142 μg/mL) but higher than for anidulafungin (0.064 μg/mL, 0.059 μg/mL). Thus, the in vitro activity of rezafungin appears comparable to anidulafungin and caspofungin against most common Candida and Aspergillus species. Rezafungin showed higher susceptibility rates against C. glabrata. Rezafungin indicates its potent activity for potential clinical application. Full article

Candida auris is an emerging multidrug-resistant and opportunistic pathogenic yeast. Whole-genome sequencing analysis has defined five major clades, each from a distinct geographic region. The current study aimed to examine the genome of the C. auris 20–1498 strain, which is the first isolate of this fungus identified in Mexico. Based on whole-genome sequencing, the draft genome was found to contain 70 contigs. It had a total genome size of 12.86 Mbp, an N50 value of 1.6 Mbp, and an average guanine-cytosine (GC) content of 45.5%. Genome annotation revealed a total of 5432 genes encoding 5515 proteins. According to the genomic analysis, the C. auris 20–1498 strain belongs to clade IV (containing strains endemic to South America). Of the two genes (ERG11 and FKS1) associated with drug resistance in C. auris, a mutation was detected in K143R, a gene located in a mutation hotspot of ERG11 (lanosterol 14-α-demethylase), an antifungal drug target. The focus on whole-genome sequencing and the identification of mutations linked to the drug resistance of fungi could lead to the discovery of new therapeutic targets and new antifungal compounds. Full article

The opportunistic fungus Candida albicans is the leading cause of invasive candidiasis in immune-compromised individuals. Drugs from the echinocandin (ECN) class, including caspofungin, are used as a first line of therapy against invasive candidiasis. The only known mechanism of clinical resistance to ECNs is point mutations in the FKS1 gene, which encodes the drug target. However, many clinical isolates developed decreased ECN susceptibilities in the absence of resistance-associated FKS1 mutations. We have identified 15 C. albicans genes that contribute to decreased drug susceptibility. We explored the expression of these 15 genes in clinical isolates with different levels of ECN susceptibility. We found that these 15 genes are expressed in clinical isolates with or without FKS1 mutations, including those strains that are less susceptible to ECNs. In addition, FKS1 expression was increased in such less susceptible isolates compared to highly susceptible isolates. Similarities of gene expression patterns between isolates with decreased ECN susceptibilities in the absence of FKS1 mutations and clinically resistant isolates with mutations in FKS1 suggest that clinical isolates with decreased ECN susceptibilities may be a precursor to development of resistance. Full article

Commercial tests are often employed in clinical microbiology laboratories for antifungal susceptibility testing of filamentous fungi. Method-dependent epidemiological cutoff values (ECVs) have been defined in order to detect non-wild-type (NWT) isolates harboring resistance mechanisms. We reviewed the literature in order to find studies where commercial methods were used to evaluate for in vitro susceptibility of filamentous fungi and assess their ability to detect NWT isolates according to the available ECVs. Data were found for the gradient concentration strips Etest and MIC Test Strips (MTS), broth microdilution Sensititre YeastOne (SYO), Micronaut-AM and the agar dilution VIPcheck assays. Applying itraconazole, voriconazole and posaconazole Etest ECVs for A. fumigatus, Etest was able to detect 90.3% (84/93), 61.2% (90/147) and 86% (31/36) of isolates with known cyp51A mutations, respectively. Moreover, Etest also was able to detect 3/3 fks mutants using caspofungin ECVs and 2/3 micafungin mutant isolates. Applying the voriconazole and posaconazole SYO ECVs, 57.7% (67/116) and 100% (47/47) of mutants with known cyp51A substitutions were classified as NWT, respectively. VIPcheck detected 90.3% (159/176), 80.1% (141/176) and 66% (141/176)of mutants via itraconazole, voriconazole and posaconazole, respectively, whereas Micronaut-AM detected 88% (22/25). In conclusion, Etest posaconazole and itraconazole, as well as micafungin and caspofungin ECVs, detected A. fumigatus mutants. On the other hand, while the posaconazole SYO ECV was able to detect cyp51A mutants, similar data were not observed with the SYO voriconazole ECV. Full article

Candida auris is a recently emerged human fungal pathogen that has posed a significant threat to public health. Since its first identification in 2009, this fungus has caused nosocomial infections in over 47 countries across all inhabited continents. As of May 2023, the whole-genome sequences of over 4000 strains have been reported and a diversity of mutations, including in genes known to be associated with drug resistance in other human fungal pathogens, have been described. Among them, 387 strains contained antifungal-susceptibility information for which different methods might be used depending on the drugs and/or investigators. In most reports on C. auris so far, the number of strains analyzed was very small, from one to a few dozen, and the statistical significance of the relationships between these genetic variants and their antifungal susceptibilities could not be assessed. In this study, we conducted genome-wide association studies on individual clades based on previously published C. auris isolates to investigate the statistical association between genomic variants and susceptibility differences to nine antifungal drugs belonging to four major drug categories: 5-fluorocytosine, amphotericin B, fluconazole, voriconazole, itraconazole, posaconazole, anidulafungin, caspofungin, and micafungin. Due to the small sample sizes for Clades II, V, and VI, this study only assessed Clades I, III, and IV. Our analyses revealed 15 single nucleotide polymorphisms (SNPs) in Clade I (10 in coding and 5 in noncoding regions), 24 SNPs in Clade III (11 in coding and 13 in noncoding regions), and 13 SNPs in clade IV (10 in coding and 3 in noncoding regions) as statistically significantly associated with susceptibility differences to one or more of the nine antifungal drugs. While four SNPs in genes encoding lanosterol 14-α-demethylase (ERG11) and the catalytic subunit of 1,3-beta-D-glucan synthase (FKS1) were shared between clades, including the experimentally confirmed Ser639Phe/Pro missense substitutions in FKS1 for echinocandin resistance, most of the identified SNPs were clade specific, consistent with their recent independent origins. Interestingly, the majority of the antifungal resistance-associated SNPs were novel, and in genes and intergenic regions that have never been reported before as associated with antifungal resistance. While targeted study is needed to confirm the role of each novel SNP, the diverse mechanisms of drug resistance in C. auris revealed here indicate both challenges for infection control and opportunities for the development of novel antifungal drugs against this and other human fungal pathogens. Full article

Non-albicans Candida infections have recently gained worldwide attention due to their intrinsic resistance to different antifungal agents and the limited therapeutic options for treating them. Although the Candida parapsilosis complex is reported to be the second or third most prevalent Candida spp., little information is available on the prevalence of antifungal resistance along with genotyping of the C. parapsilosis complex. In this study, we aimed to evaluate the prevalence of antifungal resistance, the genetic basis of such resistance, and the genotyping of C. parapsilosis complex isolates that were recovered from hospitalized patients in Japan from 2005 to 2019. Our results indicated that, with the exception of one single C. metapsilosis isolate that was dose-dependently susceptible to fluconazole, all other isolates were susceptible or showed wild phenotypes to all tested antifungals, including azoles, echinocandins, amphotericin B, and flucytosine. Molecular analyses for azole and echinocandin resistance via evaluating ERG11 mutation and FKS1 hotspot one (HS1) and hotspot two (HS2) mutations, respectively, confirmed the phenotypic results. Genotyping of our isolates confirmed that they belong to 53 different but closely related genotypes, with a similarity percentage of up to 90%. Our results are of significant concern, since understanding the genetic basis of echinocandin resistance in the C. parapsilosis complex as well their genotyping is essential for directing targeted therapy, identifying probable infection sources, and developing strategies for overcoming epidemic spread. Full article

Candidiasis is a highly pervasive infection posing major health risks, especially for immunocompromised populations. Pathogenic Candida species have evolved intrinsic and acquired resistance to a variety of antifungal medications. The primary goal of this literature review is to summarize the molecular mechanisms associated with antifungal resistance in Candida species. Resistance can be conferred via gain-of-function mutations in target pathway genes or their transcriptional regulators. Therefore, an overview of the known gene mutations is presented for the following antifungals: azoles (fluconazole, voriconazole, posaconazole and itraconazole), echinocandins (caspofungin, anidulafungin and micafungin), polyenes (amphotericin B and nystatin) and 5-fluorocytosine (5-FC). The following mutation hot spots were identified: (1) ergosterol biosynthesis pathway mutations (ERG11 and UPC2), resulting in azole resistance; (2) overexpression of the efflux pumps, promoting azole resistance (transcription factor genes: tac1 and mrr1; transporter genes: CDR1, CDR2, MDR1, PDR16 and SNQ2); (3) cell wall biosynthesis mutations (FKS1, FKS2 and PDR1), conferring resistance to echinocandins; (4) mutations of nucleic acid synthesis/repair genes (FCY1, FCY2 and FUR1), resulting in 5-FC resistance; and (5) biofilm production, promoting general antifungal resistance. This review also provides a summary of standardized inhibitory breakpoints obtained from international guidelines for prominent Candida species. Notably, N. glabrata, P. kudriavzevii and C. auris demonstrate fluconazole resistance. Full article

There is growing interest in the molecular surveillance of the Respiratory Syncytial Virus and the monitorization of emerging mutations that could impair the efficacy of antiviral prophylaxis and treatments. A simple, scalable protocol for viral nucleic acid enrichment could improve the surveillance of RSV. We developed a protocol for RSV-A and B amplification based on the Illumina CovidSeq workflow using an RSV primer panel. A total of 135 viral genomes were sequenced from nasopharyngeal samples through the optimization steps of this panel, while an additional 15 samples were used to test the final version. Full coverage of the G gene and over 95% of the coverage of the F gene, the target of the available RSV antivirals or monoclonal antibodies, were obtained. The F:K68N mutation, associated with decreased nirsevimab activity, was detected in our facility. Additionally, phylogenetic analysis showed several sublineages in the 2022–2023 influenza season in Europe. Our protocol allows for a simple and scalable simultaneous amplification of the RSV-A and B whole genome, increasing the yield of RSV sequencing and reducing costs. Its application would allow the world to be ready for the detection of arising mutations in relation to the widespread use of nirsevimab for RSV prevention. Full article

Mutations in the FKBP14 gene encoding the endoplasmic reticulum resident collagen-related proline isomerase FK506 binding protein 22 kDa (FKBP22) result in kyphoscoliotic Ehlers–Danlos Syndrome (EDS), which is characterized by a broad phenotypic outcome. A plausible explanation for this outcome is that FKBP22 participates in the biosynthesis of subsets of collagen types: FKBP22 selectively binds to collagens III, IV, VI, and X, but not to collagens I, II, V, and XI. However, these binding mechanisms have never been explored, and they may underpin EDS subtype heterogeneity. Here, we used collagen Toolkit peptide libraries to investigate binding specificity. We observed that FKBP22 binding was distributed along the collagen helix. Further, it (1) was higher on collagen III than collagen II peptides and it (2) was correlated with a positive peptide charge. These findings begin to elucidate the mechanism by which FKBP22 interacts with collagen. Full article

Echinocandins, used for the prevention and treatment of invasive fungal infections, have led to a rise in breakthrough infections caused by resistant Candida species. Among these species, those belonging to the Candida haemulonii complex are rare multidrug-resistant (MDR) yeasts that are frequently misidentified but have emerged as significant healthcare-associated pathogens causing invasive infections. The objectives of this study were to investigate the evolutionary pathways of echinocandin resistance in C. haemulonii by identifying mutations in the FKS1 gene and evaluating the impact of resistance on fitness. After subjecting a MDR clinical isolate of C. haemulonii (named Ch4) to direct selection using increasing caspofungin concentrations, we successfully obtained an isolate (designated Ch4′r) that exhibited a high level of resistance, with MIC values exceeding 16 mg/L for all tested echinocandin drugs (caspofungin, micafungin, and anidulafungin). Sequence analysis revealed a specific mutation in the resistant Ch4′r strain, leading to an arginine-histidine amino acid substitution (R1354H), occurring at the G4061A position of the HS2 region of the FKS1 gene. Compared to the wild-type strain, Ch4′r exhibited significantly reduced growth proliferation, biofilm formation capability, and phagocytosis ratio, indicating a decrease in fitness. Transmission electron microscopy analysis revealed alterations in cell wall components, with a notable increase in cell wall thickness. The resistant strain also exhibited higher amounts (2.5-fold) of chitin, a cell wall-located molecule, compared to the wild-type strain. Furthermore, the resistant strain demonstrated attenuated virulence in the Galleria mellonella larval model. The evolved strain Ch4′r maintained its resistance profile in vivo since the treatment with either caspofungin or micafungin did not improve larval survival or reduce the fungal load. Taken together, our findings suggest that the acquisition of pan-echinocandin resistance occurred rapidly after drug exposure and was associated with a significant fitness cost in C. haemulonii. This is particularly concerning as echinocandins are often the first-line treatment option for MDR Candida species. Full article

Drugs from the echinocandin (ECN) class are now recommended ‘front-line’ treatments of infections caused by a prevailing fungal pathogen, C. albicans. However, the increased use of ECNs is associated with a rising resistance to ECNs. As the acquisition of ECN resistance in C. albicans is viewed as a multistep evolution, determining factors that are associated with the decreased ECN susceptibility is of importance. We have recently identified two cohorts of genes that are either up- or downregulated in concert in order to control remodeling of cell wall, an organelle targeted by ECNs, in laboratory mutants with decreased ECN susceptibility. Here, we profiled the global DNA sequence of four of these adapted mutants in search of DNA changes that are associated with decreased ECN susceptibility. We find a limited number of 112 unique mutations representing two alternative mutational pathways. Approximately half of the mutations occurred as hotspots. Approximately half of mutations and hotspots were shared by ECN-adapted mutants despite the mutants arising as independent events and differing in some of their phenotypes, as well as in condition of chromosome 5. A total of 88 mutations are associated with 43 open reading frames (ORFs) and occurred inside of an ORF or within 1 kb of an ORF, predominantly as single-nucleotide substitution. Mutations occurred more often in the 5′-UTR than in the 3′-UTR by a 1.67:1 ratio. A total of 16 mutations mapped to eight genomic features that were not ORFs: Tca4-4 retrotransposon; Tca2-7 retrotransposon; lambda-4a long terminal repeat; mu-Ra long terminal repeat; MRS-7b Major Repeat Sequence; MRS-R Major Repeat Sequence; RB2-5a repeat sequence; and tL (CAA) leucine tRNA. Finally, eight mutations are not associated with any ORF or other genomic feature. Repeated occurrence of single-nucleotide substitutions in non-related drug-adapted mutants strongly indicates that these DNA changes are accompanying drug adaptation and could possibly influence ECN susceptibility, thus serving as factors facilitating evolution of ECN drug resistance due to classical mutations in FKS1. Full article

The carriage of Candida albicans in children’s oral cavities is associated with a higher risk for early childhood caries, so controlling this fungus in early life is essential for preventing caries. In a prospective cohort of 41 mothers and their children from 0 to 2 years of age, this study addressed four main objectives: (1) Evaluate in vitro the antifungal agent susceptibility of oral Candida isolates from the mother-child cohort; (2) compare Candida susceptibility between isolates from the mothers and children; (3) assess longitudinal changes in the susceptibility of the isolates collected between 0 and 2 years; and (4) detect mutations in C. albicans antifungal resistance genes. Susceptibility to antifungal medications was tested by in vitro broth microdilution and expressed as the minimal inhibitory concentration (MIC). C. albicans clinical isolates were sequenced by whole genome sequencing, and the genes related to antifungal resistance, ERG3, ERG11, CDR1, CDR2, MDR1, and FKS1, were assessed. Four Candida spp. (n = 126) were isolated: C. albicans, C. parapsilosis, C. dubliniensis, and C. lusitaniae. Caspofungin was the most active drug for oral Candida, followed by fluconazole and nystatin. Two missense mutations in the CDR2 gene were shared among C. albicans isolates resistant to nystatin. Most of the children’s C. albicans isolates had MIC values similar to those from their mothers, and 70% remained stable on antifungal medications from 0 to 2 years. For caspofungin, 29% of the children’s isolates showed an increase in MIC values from 0 to 2 years. Results of the longitudinal cohort indicated that clinically used oral nystatin was ineffective in reducing the carriage of C. albicans in children; novel antifungal regimens in infants are needed for better oral yeast control. Full article

Outbreaks of invasive infections, with high mortality rates, caused by multidrug-resistant Candida auris have been reported worldwide. Although hotspot mutations in FKS1 are an established cause of echinocandin resistance, the actual contribution of these mutations to echinocandin resistance remains unknown. Here, we sequenced the FKS1 gene of a caspofungin-resistant clinical isolate (clade I) and identified a novel resistance mutation (G4061A inducing R1354H). We applied the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to generate a recovered strain (H1354R) in which only this single nucleotide mutation was reverted to its wild-type sequence. We also generated mutant strains with only the R1354H mutation introduced into C. auris wild-type strains (clade I and II) and analyzed their antifungal susceptibility. Compared to their parental strains, the R1354H mutants exhibited a 4- to 16-fold increase in caspofungin minimum inhibitory concentration (MIC) while the H1354R reverted strain exhibited a 4-fold decrease in caspofungin MIC. In a mouse model of disseminated candidiasis, the in vivo therapeutic effect of caspofungin was more closely related to the FKS1 R1354H mutation and the virulence of the strain than its in vitro MIC. The CRISPR-Cas9 system could thus aid in elucidating the mechanism underlying drug resistance in C. auris. Full article