Diagnostic Allele-Specific PCR for the Identification of Candida auris Clades.
Ontology highlight
ABSTRACT:
SUBMITTER: Carolus H
PROVIDER: S-EPMC8471779 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Candida auris is a multidrug-resistant fungus against which in some clinical situations amphotericin B (AMB) remains the alternative or first line drug. We compared daily 1 mg/kg of AMB efficacy in a neutropenic murine bloodstream infection model against 10 isolates representing four C. auris clades (South Asian n = 2; East Asian n = 2; South African n = 2; South American n = 4; two of which were of environmental origin). Five days of AMB treatment significantly increased the survival rates in mice infected with isolates of the East Asian clade, and 1 isolate each from the South African and South American clades (originated from bloodstream), but not in mice infected with the South Asian and 2 environmental isolates from the South American clades. AMB treatment decreased the fungal burden in mice infected with the 2 isolates each from East Asian and South African, and 1 out of 2 bloodstream isolates from South American clades in the hearts (p < 0.01), kidneys (p < 0.01) and brain (p < 0.05). AMB treatment, regardless of clades, significantly decreased colony forming units in the urine at day 3. However, histopathological examination in AMB-treated mice revealed large aggregates of yeast cells in the kidneys and hearts, and focal lesions in the cerebra and cerebelli, regardless of precise C. auris clade. Our clade-specific data confirm that the efficacy of AMB against C. auris is weak, explaining the therapeutic failures in clinical situations. Our results draw attention to the necessity to maximize the killing at the start of treatment to avoid later complications in the heart and central nervous system.
| S-EPMC9144575 | biostudies-literature
Project description:Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multi-allelic S-locus. Information about the S-genotypes is important for breeding and the selection of pollen donors for fruit production. Rapid and reliable S-genotype identification system is necessary for efficient breeding of new cultivars in Japanese pear. We designed S allele-specific PCR primer pairs for ten previously reported S-RNase alleles (S (1)-S (9) and S (k)) as simple and reliable method. Specific nucleotide sequences were chosen to design the primers to amplify fragments of only the corresponding S alleles. The developed primer pairs were evaluated by using homozygous S-genotypes (S (1)/S (1)-S (9)/S (9) and S (4sm)/S (4sm)) and 14 major Japanese pear cultivars, and found that S allele-specific primer pairs can identify S-genotypes effectively. The S allele-specific primer pairs developed in this study will be useful for efficient S-genotyping and for marker-assisted selection in Japanese pear breeding programs.
| S-EPMC4482170 | biostudies-literature
Project description:BackgroundLeishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.MethodsA probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.ResultsThis new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.ConclusionsOur assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.
| S-EPMC9164491 | biostudies-literature
Project description:Background and purposeCandida auris is an emerging multidrug-resistant pathogen. The identification of this species with the conventional phenotypic or biochemical mycological methods may lead to misidentification. Molecular-based species-specific identification methods such as quantitative real-time polymerase chain reaction (qPCR) facilitate a more reliable identification of C. auris than mycological methods. Regarding this, the present study aimed to develop a hydrolysis probe-based qPCR assay for the rapid, accurate identification of C. auris.Materials and methodsThe internal transcribed spacer 2 regions in the nuclear ribosomal DNA of C. auris and other related yeasts were assayed to find a specific PCR target for C. auris. A 123-base-pair target was selected, and primers and a probe were designed for hydrolysis probe-based real-time PCR with TaqMan chemistry. Ten-fold serial dilutions of C. auris ranging from 106 to 100 CFU/mL were prepared to establish a standard curve to quantify the yeast.ResultsThe qPCR assay was able to identify and quantify C. auris with a detection limit of 1 C. auris CFU per reaction. Specificity was confirmed by the non-amplification of the sequences belonging to other Candida species, yeasts, molds, bacteria, or human DNAs. The standard curve of the assay showed a highly significant linearity between threshold values and dilution rates (R2=0.99; slope=-3.42).ConclusionThe applied qPCR assay facilitated the rapid and accurate identification and quantification of emerging opportunistic C. auris. Therefore, considering the promising test validation results, we succeeded to develop a rapid and accurate hydrolysis probe- based qPCR assay for the screening and identification of C. auris.
| S-EPMC8018816 | biostudies-literature
Project description:Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii, Candida haemulonii, and Candida lusitaniae Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species.
| S-EPMC5527423 | biostudies-other
Project description:Antimicrobial resistance is a global health crisis to which pathogenic fungi make a substantial contribution. The human fungal pathogen C. auris is of particular concern due to its rapid spread across the world and its evolution of multidrug resistance. Fluconazole failure in C. auris has been recently attributed to antifungal "tolerance". Tolerance is a phenomenon whereby a slow-growing subpopulation of tolerant cells, which are genetically identical to susceptible cells, emerges during drug treatment. We use microbroth dilution and disk diffusion assays, together with image analysis, to investigate antifungal tolerance in C. auris to all three classes of antifungal drugs used to treat invasive candidiasis. We find that (1) C. auris is tolerant to several common fungistatic and fungicidal drugs, which in some cases can be detected after 24 h, as well as after 48 h, of antifungal drug exposure; (2) the tolerant phenotype reverts to the susceptible phenotype in C. auris; and (3) combining azole, polyene, and echinocandin antifungal drugs with the adjuvant chloroquine in some cases reduces or eliminates tolerance and resistance in patient-derived C. auris isolates. These results suggest that tolerance contributes to treatment failure in C. auris infections for a broad range of antifungal drugs, and that antifungal adjuvants may improve treatment outcomes for patients infected with antifungal-tolerant or antifungal-resistant fungal pathogens.
| S-EPMC10045952 | biostudies-literature
Project description:Candida auris is a multidrug-resistant pathogen against which echinocandins are the drug of choice. However, information on how the chitin synthase inhibitor nikkomycin Z influences the killing activities of echinocandins against C. auris is currently lacking. We determined the killing activities of anidulafungin and micafungin (0.25, 1, 8, 16 and 32 mg/L each) with and without nikkomycin Z (8 mg/L) against 15 isolates representing four C. auris clades (South Asian n = 5; East Asian n = 3; South African n = 3; South American n = 4, two of which were of environmental origin). Two and one isolates from the South Asian clade harbored mutations in the hot-spot 1 (S639Y and S639P) and 2 (R1354H) regions of the FKS1 gene, respectively. The anidulafungin, micafungin and nikkomycin Z MIC ranges were 0.015-4, 0.03-4 and 2->16 mg/L, respectively. Anidulafungin and micafungin alone exerted weak fungistatic activity against wild-type isolates and the isolate with a mutation in the hot-spot 2 region of FKS1 but was ineffective against the isolates with a mutation in the hot-spot 1 region. The nikkomycin Z killing curves were always similar to their respective controls. Twenty-two of sixty (36.7%) anidulafungin plus nikkomycin Z and twenty-four of sixty (40%) micafungin plus nikkomycin Z combinations produced at least 100-fold decreases in the CFUs (synergy), with a 41.7% and 20% fungicidal effect, respectively, against wild-type isolates. Antagonism was never observed. Similar results were found with the isolate with a mutation in hot-spot 2 of FKS1, but the combinations were ineffective against the two isolates with prominent mutations in hot-spot 1 of FKS1. The simultaneous inhibition of β-1,3 glucan and chitin synthases in wild-type C. auris isolates produced significantly greater killing rates than either drug alone. Further studies are warranted to verify the clinical efficacy of echinocandin plus nikkomycin Z combinations against echinocandin susceptible C. auris isolates.
| S-EPMC10222763 | biostudies-literature
Project description:Candida nivariensis is a recently described pathogenic yeast closely related to Candida glabrata. We developed a specific set of oligonucleotide primers based on the internal transcribed spacer regions of the rRNA gene for the rapid identification of C. nivariensis. PCR with these primers amplified a 206-bp amplicon from C. nivariensis.
| S-EPMC1317211 | biostudies-literature
Project description:Candida auris is an emerging fungal pathogen of rising concern due to global spread, the ability to cause healthcare-associated outbreaks, and antifungal resistance. Genomic analyses revealed that early contemporaneously detected cases of C. auris were geographically stratified into four major clades. While Clades I, III, and IV are responsible for ongoing outbreaks of invasive and multidrug-resistant infections, Clade II, also termed the East Asian clade, consists primarily of cases of ear infection, is often susceptible to all antifungal drugs, and has not been associated with outbreaks. Here, we generate chromosome-level assemblies of twelve isolates representing the phylogenetic breadth of these four clades and the only isolate described to date from Clade V. This Clade V genome is highly syntenic with those of Clades I, III, and IV, although the sequence is highly divergent from the other clades. Clade II genomes appear highly rearranged, with translocations occurring near GC-poor regions, and large subtelomeric deletions in most chromosomes, resulting in a substantially different karyotype. Rearrangements and deletion lengths vary across Clade II isolates, including two from a single patient, supporting ongoing genome instability. Deleted subtelomeric regions are enriched in Hyr/Iff-like cell-surface proteins, novel candidate cell wall proteins, and an ALS-like adhesin. Cell wall proteins from these families and other drug-related genes show clade-specific signatures of selection in Clades I, III, and IV. Subtelomeric dynamics and the conservation of cell surface proteins in the clades responsible for global outbreaks causing invasive infections suggest an explanation for the different phenotypes observed between clades.
| S-EPMC8128392 | biostudies-literature
Project description:Candida auris, a multidrug-resistant human fungal pathogen that causes outbreaks of invasive infections, emerged as four distinct geographical clades. Previous studies identified genomic and proteomic differences in nutrient utilization on comparison to Candida albicans, suggesting that certain metabolic features may contribute to C. auris emergence. Since no high-throughput clade-specific metabolic characterization has been described yet, we performed a phenotypic screening of C. auris strains from all 4 clades on 664 nutrients, 120 chemicals, and 24 stressors. We identified common and clade- or strain-specific responses, including the preferred utilization of various dipeptides as nitrogen source and the inability of the clade II isolate AR 0381 to withstand chemical stress. Further analysis of the metabolic properties of C. auris isolates showed robust growth on intermediates of the tricarboxylic acid cycle, such as citrate and succinic and malic acids. However, there was reduced or no growth on pyruvate, lactic acid, or acetate, likely due to the lack of the monocarboxylic acid transporter Jen1, which is conserved in most pathogenic Candida species. Comparison of C. auris and C. albicans transcriptomes of cells grown on alternative carbon sources and dipeptides as a nitrogen source revealed common as well as species-unique responses. C. auris induced a significant number of genes with no ortholog in C. albicans, e.g., genes similar to the nicotinic acid transporter TNA1 (alternative carbon sources) and to the oligopeptide transporter (OPT) family (dipeptides). Thus, C. auris possesses unique metabolic features which could have contributed to its emergence as a pathogen. IMPORTANCE Four main clades of the emerging, multidrug-resistant human pathogen Candida auris have been identified, and they differ in their susceptibilities to antifungals and disinfectants. Moreover, clade- and strain-specific metabolic differences have been identified, but a comprehensive overview of nutritional characteristics and resistance to various stressors is missing. Here, we performed high-throughput phenotypic characterization of C. auris on various nutrients, stressors, and chemicals and obtained transcriptomes of cells grown on selected nutrients. The generated data sets identified multiple clade- and strain-specific phenotypes and induction of C. auris-specific metabolic genes, showing unique metabolic properties. The presented work provides a large amount of information for further investigations that could explain the role of metabolism in emergence and pathogenicity of this multidrug-resistant fungus.
| S-EPMC10269459 | biostudies-literature