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PURPOSE: The identification of anaerobes, Mycobacterium and Nocardia species, and moulds by MALDI-TOF-MS remains a challenge. This study aimed to evaluate the performance of MALDI-TOF in the identification of these organisms. METHODS: A total of 382 strains, comprising 128 (33.5 %) anaerobes, 126(33.0 %) mycobacterial, 113(29.6 %), mycelial fungi, and 15(3.9 %) Nocardia species were evaluated by VITEK MS Version 3.0. The results were compared with the identification of the isolates by DNA sequence analysis. The DNA sequences used for analysis were the 16S rRNA for anaerobic bacteria, hsp65 gene for mycobacteria, whereas both 16S rRNA and hsp65 gene for Nocardia species, and internal transcribed spacer (ITS) and 28S rRNA gene's D1/D2 regions of fungi. RESULTS: The VITEK-MS accurately identified 78.3 % (299/382) of the strains at the species, and 9.4 % (36/382) at the genus level. Misidentifications were observed in 3.9 % (15/382) isolates. Of isolates tested, 8.4 % (32/382) were not identified by the system, and 7.06 % (27/382) were not included in the IVD database. CONCLUSION: An upgraded VITEK MS V3.0 database provides reasonably accurate and rapid identification of clinically relevant anaerobes, mycobacteria, Nocardia species, and moulds to the species level.
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BACKGROUND: Over the past decades, the increasing incidence of recurrent dermatophytosis associated with terbinafine-resistant Trichophyton has posed a serious challenge in management of dermatophytosis. Independent reports of failure of treatment and high minimum inhibitory concentrations (MIC) of antifungals are available, but data correlating MIC and clinical outcomes is still sparse. Therefore, the present study was conducted to evaluate the outcomes of systemic treatment of dermatophytosis and its correlation with MIC of the etiological agents isolated from such patients. METHODS: Retrospective analysis of 587 consecutive patients with dermatophytosis was done from March 2017 to March 2019. Demographic and clinical details of the patients were noted, along with the results of direct microscopy and fungal culture. The isolates were identified by sequencing the internal transcribed spacer region of rDNA. Antifungal susceptibility testing was performed following the CLSI M38 protocol. Mutation in the squalene epoxidase (SE) gene was detected by DNA sequencing and ARMS-PCR. Based on the culture-positivity and prescribed systemic antifungal, patients were categorised into Group I culture-positive cases treated with systemic terbinafine and Group II culture-positive cases treated with systemic itraconazole, each for a total period of 12 weeks. RESULTS: In the present study, 477 (81.39%) were culture-positive; however, 12 weeks follow-up was available for 294 patients (Group I-157 and Group II-137) who were included for statistical analysis. In both groups [Group I-37/63 (51.4%) and Group II-14/54 (58.3%)], a better cure rate was observed if the initiation of therapy was performed within <6 months of illness. Treatment outcome revealed that if therapy was extended for 8-12 weeks, the odds of cure rate are significantly better (p < .001) with either itraconazole (Odd Ratio-15.5) or terbinafine (Odd Ratio-4.34). Higher MICs for terbinafine were noted in 41 cases (cured-18 and uncured-23) in Group I and 39 cases (cured-16 and uncured-23) in Group II. From cured (Group I-17/18; 94.4% and Group II-14/16; 87.5%) and uncured (Group I-20/23; 86.9% and Group II-21/23; 91.3%) cases had F397L mutation in the SE gene. No significant difference in cure rate was observed in patients with Trichophyton spp. having terbinafine MIC ≥ 1or <1 µg/mL (Group I-p = .712 and Group II-p = .69). CONCLUSION: This study revealed that prolonging terbinafine or itraconazole therapy for beyond 8 weeks rather than the standard 4 weeks significantly increases the cure rate. Moreover, no correlation has been observed between antifungal susceptibility and clinical outcomes. The MIC remains the primary parameter for defining antifungal activity and predicting the potency of antifungal agents against specific fungi. However, predicting therapeutic success based solely on the MIC of a fungal strain is not always reliable, as studies have shown a poor correlation between in vitro data and in vivo outcomes. To address this issue, further correlation of antifungal susceptibility testing (AFST) data with clinical outcomes and therapeutic drug monitoring is needed. It also highlights that initiation of the treatment within <6 months of illness increases cure rates and reduces recurrence. Extensive research is warranted to establish a better treatment regime for dermatophytosis.
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Antifúngicos , Itraconazol , Mutação , Esqualeno Mono-Oxigenase , Terbinafina , Tinha , Trichophyton , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Antifúngicos/uso terapêutico , Antifúngicos/farmacologia , Farmacorresistência Fúngica/genética , Itraconazol/farmacologia , Itraconazol/uso terapêutico , Testes de Sensibilidade Microbiana , Estudos Retrospectivos , Esqualeno Mono-Oxigenase/genética , Terbinafina/uso terapêutico , Terbinafina/farmacologia , Tinha/tratamento farmacológico , Tinha/microbiologia , Resultado do Tratamento , Trichophyton/efeitos dos fármacos , Trichophyton/genéticaRESUMO
Multidrug-resistant yeast Candida auris is a serious threat to public health with documented survival in various hospital niches. The dynamics of this survival benefit and its trade off with drug resistance are still unknown for this pathogen. In this study we investigate the oxidative stress response (OSR) in fluconazole-resistant C. auris and compare its relative fitness with fluconazole-susceptible strains. A total of 351 C. auris clinical isolates (61 fluconazole-susceptible and 290 fluconazole-resistant) were screened for stress tolerance by spot assay and 95.08 % fluconazole-susceptible isolates were hyper-resistant to oxidative stress while majority (94.5 %) fluconazole-resistant isolates had lower oxidative tolerance. Expression of Hog1 and Cta1 gene transcript levels and cellular catalase levels were significantly higher in fluconazole-susceptible isolates and a corresponding higher intracellular reactive oxygen species level (iROS) was accumulated in the fluconazole-resistant isolates. Biofilm formation and cell viability under oxidative stress revealed higher biofilm formation and better viability in fluconazole-susceptible isolates. Fluconazole-resistant isolates had higher basal cell wall chitin. On comparison of virulence, the % cytotoxicity in A549 cell line was higher in fluconazole-susceptible isolates and the median survival of the infected larvae in G. mellonella infection model was higher in fluconazole-resistant (5; IQR:4.5-5 days) vs. fluconazole-susceptible C. auris (2; IQR:1.5-2.5 days). All organisms evolve with changes in their environmental conditions, to ensure an optimal balance between proliferation and survival. Development of tolerance to a certain kind of stress example antifungal exposure in yeast can leads to a compensatory decrease in tolerance for other stresses. This study provides useful insights into the comparative fitness and antifungal susceptibility trade off in C. auris. We report a negative association between H2O2 tolerance and fluconazole susceptibility. Using in-vitro cell cytotoxicity and in-vivo survival assays we also demonstrate the higher virulence potential of fluconazole-susceptible C. auris isolates corroborating the negative correlation between susceptibility and pathogen survival or virulence. These findings could also be translated to clinical practice by investigating the possibility of using molecules targeting stress response and fitness regulating pathways for management of this serious infection.
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Acrophialophora is implicated in superficial and invasive infections, especially in immunosuppressed individuals. The present study was undertaken to provide clinical, microbiological, phylogenetic, and antifungal susceptibility testing (AFST) profile of Acrophialophora isolated from India. All the isolates identified as Acrophialophora species at the National Culture Collection for Pathogenic Fungi, Chandigarh, India were revived. Phenotypic and molecular characterization was performed, followed by temperature studies, scanning electron microscopy (SEM), and AFST. We also performed systematic review of all the cases of Acrophialophora species reported till date. A total of nine isolates identified as Acrophialophora species were identified by molecular method as A. fusispora (n = 8) and A. levis (n = 1), from brain abscess (n = 4), respiratory tract (n = 3), and corneal scraping (n = 2). All patients but two had predisposing factors/co-morbidities. Acrophialophora was identified as mere colonizer in one. Temperature studies and SEM divulged variation between both species. Sequencing of the internal transcribed spacer ribosomal DNA and beta-tubulin loci could distinguish species, while the LSU ribosomal DNA locus could not. AFST showed the lowest minimum inhibitory concentrations (MICs) for triazoles and the highest for echinocandins. Systematic literature review revealed 16 cases (11 studies), with ocular infections, pulmonary and central nervous system infections, and A. fusispora was common species. All the patients except three responded well. High MICs were noted for fluconazole, micafungin, and caspofungin. This is the first study delineating clinical, phenotypic, and genotypic characteristics of Acrophialophora species from India. The study highlights microscopic differences between both species and emphasizes the role of molecular methods in precise identification. Triazoles appear to be the most effective antifungals for managing patients.
We describe clinical, phenotypic, and genotypic characteristics of Acrophialophora species. This species causes mild infection to fatal infection in immunosuppressed individuals. Triazoles are effective in treating such infections.
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Antifúngicos , Testes de Sensibilidade Microbiana , Micoses , Filogenia , Índia , Humanos , Antifúngicos/farmacologia , Adulto , Masculino , Micoses/microbiologia , Feminino , Pessoa de Meia-Idade , Ascomicetos/efeitos dos fármacos , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Ascomicetos/classificação , DNA Fúngico/genética , Análise de Sequência de DNA , DNA Espaçador Ribossômico/genética , Microscopia Eletrônica de Varredura , Fenótipo , Tubulina (Proteína)/genética , Idoso , Adulto Jovem , CriançaRESUMO
During surge of COVID-19-associated mucormycosis (CAM), we identified five cases of CAM where two different species of Mucorales were isolated. All had history of diabetes mellitus and presented with clinical features suggesting rhino-orbital mucormycosis. The patients grew different species from their nasal scraping/biopsy samples, Rhizopus arrhizus, R. homothallicus (n = 2); R. homothallicus, Lictheimia corymbifera (n = 1); R. arrhizus, Mucor spp (n = 1); and L. corymbifera, Apophysomyces variabilis (n = 1). All patients underwent surgical and medical (liposomal amphotericin B) treatment. All, except one growing A. variabilis and L. corymbifera survived. Mixed infection by more than one Mucorales in CAM is unique and warrants epidemiological investigation.
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COVID-19 , Mucorales , Mucormicose , Mucormicose/diagnóstico , Mucormicose/microbiologia , Humanos , Mucorales/isolamento & purificação , Mucorales/classificação , Masculino , Pessoa de Meia-Idade , COVID-19/complicações , Feminino , Antifúngicos/uso terapêutico , Anfotericina B/uso terapêutico , Adulto , SARS-CoV-2 , IdosoRESUMO
BACKGROUND: Due to a delay in diagnosis by conventional techniques and high mortality, the development of a standardised and rapid non-culture-based technique is an unmet need in pulmonary, gastrointestinal, and disseminated forms of mucormycosis. Though limited studies have been conducted for molecular diagnosis, there are no established serologic tests for this highly fatal infection. OBJECTIVE: To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis. METHODS: We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 µg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample. RESULTS: Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity. CONCLUSION: The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.
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Anticorpos Antifúngicos , Antígenos de Fungos , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G , Imunoglobulina M , Mucormicose , Rhizopus , Sensibilidade e Especificidade , Testes Sorológicos , Mucormicose/diagnóstico , Mucormicose/microbiologia , Mucormicose/imunologia , Humanos , Rhizopus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Antígenos de Fungos/imunologia , Antígenos de Fungos/análise , Testes Sorológicos/métodos , Anticorpos Antifúngicos/sangue , Imunoglobulina M/sangue , Imunoglobulina G/sangue , Feminino , Masculino , Pessoa de Meia-IdadeRESUMO
Candida tropicalis is a human pathogen and one of the most prevalent non-Candida albicans Candida (NCAC) species causing invasive infections. Azole antifungal resistance in C. tropicalis is also gradually increasing with the increasing incidence of infections. The pathogenic success of C. tropicalis depends on its effective response in the host microenvironment. To become a successful pathogen, cellular metabolism, and physiological status determine the ability of the pathogen to counter diverse stresses inside the host. However, to date, limited knowledge is available on the impact of carbon substrate metabolism on stress adaptation and azole resistance in C. tropicalis. In this study, we determined the impact of glucose, fructose, and sucrose as the sole carbon source on the fluconazole resistance and osmotic (NaCl), oxidative (H2O2) stress adaptation in C. tropicalis clinical isolates. We confirmed that the abundance of carbon substrates influences or increases drug resistance and osmotic and oxidative stress tolerance in C. tropicalis. Additionally, both azole-resistant and susceptible isolates showed similar stress adaptation phenotypes, confirming the equal efficiency of becoming successful pathogens irrespective of drug susceptibility profile. To the best of our knowledge, our study is the first on C. tropicalis to demonstrate the direct relation between carbon substrate metabolism and stress tolerance or drug resistance.
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Antifúngicos , Candida tropicalis , Carbono , Farmacorresistência Fúngica , Fluconazol , Testes de Sensibilidade Microbiana , Estresse Oxidativo , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/fisiologia , Antifúngicos/farmacologia , Humanos , Fluconazol/farmacologia , Carbono/metabolismo , Candidíase/microbiologia , Pressão Osmótica , Glucose/metabolismo , Sacarose/metabolismo , Sacarose/farmacologia , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Frutose/metabolismo , Frutose/farmacologia , Estresse FisiológicoRESUMO
We utilized linear and 2D infrared spectroscopy to analyze the carbonyl stretching modes of small esters in different solvents. Particularly noteworthy were the distinct carbonyl spectral line shapes in aqueous solutions, prompting our investigation of the underlying factors responsible for these differences. Through our experimental and theoretical calculations, we identified the presence of the hydrogen-bond-induced Fermi resonance as the primary contributor to the varied line shapes of small esters in aqueous solutions. Furthermore, our findings revealed that the skeletal deformation mode plays a crucial role in the Fermi resonance for all small esters. Specifically, the first overtone band of the skeletal deformation mode intensifies when hydrogen bonds form with the carbonyl group of esters, whereas such coupling is rare in aprotic organic solvents. These spectral insights carry significant implications for the utilization of esters as infrared probes in both biological and chemical systems.
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Two-dimensional (2D) van der Waals (vdW) materials, including graphene, hexagonal boron nitride (hBN), and metal dichalcogenides (MCs), form the basis of modern electronics and optoelectronics due to their unique electronic structure, chemical activity, and mechanical strength. Despite many proof-of-concept demonstrations so far, to fully realize their large-scale practical applications, especially in devices, wafer-scale single crystal atomically thin highly uniform films are indispensable. In this minireview, we present an overview on the strategies and highlight recent significant advances toward the synthesis of wafer-scale single crystal graphene, hBN, and MC 2D thin films. Currently, there are five distinct routes to synthesize wafer-scale single crystal 2D vdW thin films: (i) nucleation-controlled growth by suppressing the nucleation density, (ii) unidirectional alignment of multiple epitaxial nuclei and their seamless coalescence, (iii) self-collimation of randomly oriented grains on a molten metal, (iv) surface diffusion and epitaxial self-planarization and (v) seed-mediated 2D vertical epitaxy. Finally, the challenges that need to be addressed in future studies have also been described.
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BACKGROUND: Cladosporium halotolerans is a saprobic fungus, rarely implicated in human infections. The identification is challenging due to non-specific phenotypic features. OBJECTIVE: To decipher clinical spectrum, microbiological and susceptibility profile of clinical and environmental isolates of Cladosporium halotolerans. METHOD: All the isolates identified as Cladosporium halotolerans deposited in National Culture Collection for Pathogenic Fungi (NCCPF), Postgraduate Institute of Medical Education and Research, Chandigarh, India were revived. Phenotypic and molecular characterization targeting internal transcribed spacer (ITS) region of ribosomal DNA, large subunit of ribosomal DNA (LSU; NL1 and NL4), actin (ACT) and beta-tubulin (TUB) was done. Scanning electron microscopy (SEM) was performed to determine any phenotypic variations. Antifungal susceptibility testing (AFST) was carried out for eight antifungal agents as per CLSI M38 Ed3 guidelines. We also performed systematic literature review of all the cases of Cladosporium halotolerans reported till date. RESULTS: A total of four isolates (clinical, n = 3; soil, n = 1) identified as Cladosporium halotolerans were included in the study. The clinical sites were skin, maxillary tissue and nail. All patients were apparently immunocompetent, and history of trauma was recorded in one patient. All patients improved on antifungal therapy. The cultures revealed growth of black mycelial fungus and microscopic examination demonstrated dematiaceous septate hyphae with erect conidiophores and conidia in branched acropetal chains. Based on molecular methods, all the four isolates were identified as C. halotolerans. SEM revealed no variation in length and width of the conidia, conidiophores, ramoconidium and hyphae among the isolates. All molecular targets, such as ITS region, LSU (partially sequenced), ACT and TUB were able to differentiate the isolates. Minimum inhibitory concentrations for antifungals were: triazoles (0.12-2 µg/ml), amphotericin B (4 µg/ml) and echinocandins (2-8 µg/ml). CONCLUSION: We report role of the rarely isolated dematiaceous fungus, C. halotolerans, in causing human infections. The study emphasizes the role of molecular methods in precisely identifying these species. Triazoles are more active against these black fungi compared to polyenes or echinocandins.
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Antifúngicos , Fungos , Humanos , Antifúngicos/farmacologia , Fungos/genética , Equinocandinas/farmacologia , DNA Ribossômico/genética , Triazóis , Microscopia Eletrônica de Varredura , Esporos FúngicosRESUMO
Metabolic reprogramming is a significant hallmark of cancer that promotes chemoresistance by allowing tumor tissues to adapt to changes in the tumor microenvironment caused by anticancer therapies. Hepatocellular carcinoma (HCC), one of the most common types of primary tumors, is associated with recurrent metabolic reprogramming that maximizes cancer cell growth and proliferation. Herein, we developed metformin (MET)-loaded hyaluronic acid (HA)-derived carbon dots (HA-CD-MET) by a simple and green method with no involvement of any additives. HA-CD-MET was utilized for specifically binding the CD44 receptor overexpressed in HCC and induced glutamine metabolic rewiring to inhibit HCC cell proliferation. Exposure to HA-CD-MET resulted in â¼6.5-fold better anticancer efficacy against CD44+ Hep3B cells in comparison to CD44-, HepG2, and noncancerous HEK293 cells at a very low dose of 80 µg/mL. Moreover, treatment of three-dimensional (3D) tumor spheroid model of HCC (Hep3B) with HA-CD-MET resulted in â¼4.9-fold reduction in tumor size. This improved anticancer efficacy of HA-CD-MET was attributed to the inhibition of glutaminase-1 (GLS-1), a mitochondrial enzyme that hydrolyzes glutamine into glutamate as confirmed from immunofluorescence and immunoblotting experiments. Furthermore, treatment with HA-CD-MET resulted in downregulation of glucose transporter-1 (GLUT-1) in Hep3B cells. Consequently, cancer cells were starved from essential nutrients, glutamine, and glucose, leading to the enhancement in intracellular ROS generation. This increase in intracellular ROS accumulation activated AMP-activated protein kinase (AMPK) and inhibited AKT phosphorylation, leading to cancer cell apoptosis. Thus, this study offers the targeting of metabolic reprogramming by HA-CD-MET that opens up a promising strategy for therapeutic intervention in hepatocarcinoma.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Metformina , Humanos , Carcinoma Hepatocelular/patologia , Metformina/farmacologia , Metformina/uso terapêutico , Ácido Hialurônico , Glutamina , Neoplasias Hepáticas/patologia , Carbono , Espécies Reativas de Oxigênio/metabolismo , Células HEK293 , Linhagem Celular Tumoral , Receptores de Hialuronatos/metabolismo , Microambiente TumoralRESUMO
Fluorometric sensors have gained considerable attention in various fields, including environmental monitoring, biomedical research, and clinical diagnostics. This article delineates the fabrication of an orange emitting naphthalene diimide (NDI) derivative consisting of maleimide moiety (NDI-mal) for fluorometric sensing of thiols. Spherical shaped organic nanoparticles (â¼100-150 nm) were constructed by NDI-mal in dimethyl sulfoxide (DMSO)/dimethylformamide (DMF)-water through J-type aggregation. NDI-mal displayed self-assembly driven aggregation-induced emission (AIE) through excimer formation at λem= 588 nm at fw = 99 vol % DMSO/DMF-water. Naphthyl residue at both terminals of NDI-mal facilitates intramolecular charge transfer (ICT) from the donor naphthyl residue to the acceptor NDI core. The fluorescence intensity of NDI-mal fluorescent organic nanoparticles (FONPs) got quenched in the presence of thiols due to thiol-maleimide adduct formation (Michael addition). NDI-mal FONPs selectively probed thiol functionalized small molecules (4-aminothiophenol), biomolecules (glutathione (GSH)), and proteins (reduced BSA) with high sensitivity having a limit of detection of 15.3 nM, 6.0 nM, and 9.2 ng/mL, respectively. Importantly, thiol sensing was selective against analogous small molecules, biomolecules, and proteins devoid of thiol moieties. Cellular imaging demonstrated selective diagnosis of cancer cells by NDI-mal FONPs through quenching of its emission upon interaction with thiols in B16F10 cells due to the high abundance of GSH in cancer cells compared to NIH3T3 cells. NDI-mal FONPs emitted their native fluorescence inside cells subjected to reactive oxygen species mediated thiol oxidation via Fenton's reaction. Notably, GSH-maleimide adduct formation by NDI-mal FONPs displayed notable therapeutic efficacy against cancer cells having â¼2.4-fold higher killing of B16F10 in comparison to NIH3T3 cells possibly through oxidative stress induced apoptosis owing to the depletion in the GSH level. Thus, NDI-mal AIE-gen successfully emerged as a selective and sensitive probe toward thiols through thiol-maleimide click chemistry with therapeutic ability against cancer cells in the absence of systematic intervention.
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Dimetil Sulfóxido , Compostos de Sulfidrila , Animais , Camundongos , Células NIH 3T3 , Compostos de Sulfidrila/química , Maleimidas/química , Proteínas , Corantes Fluorescentes/química , ÁguaRESUMO
Aggregation-induced emission (AIE)-based fluorescent organic nanoparticles (FONPs) with distinctive characteristics are emerging as superior sensors due to their facile fabrication, high signal-to-noise ratio, and good biocompatibility. The present article delineates the detection and analysis of the redox behavior of the protein disulfide isomerase (PDI) enzyme by exploitation of the AIE of novel naphthalimide (NI) derivatives having thiol (-SH) and disulfide (-S-S-) moieties. Self-aggregated spherical-shaped organic nanoparticles were prepared by synthesized NI-based amphiphiles (NISH, NISS, NINSS, and TNINSH) through J-type aggregation in DMSO-water (fw = 99 vol %). Naphthyl residue containing NI-derived amphiphiles (NINSS and TNINSH) exhibited AIE (blue and yellow) at 470 and 550 nm, respectively, in DMSO-water (fw = 99 vol %). NINSS and TNINSH FONPs were suitably utilized in sensing PDI through their redox nature of thiol-disulfide exchange. Fluorescence quenching of NINSS FONPs was observed due to reduction of disulfide to thiol by PDI, whereas emission intensity was progressively red-shifted and enhanced ("Dual-AIE") for TNINSH (containing ER-targeting N-tosylethylenediamine), owing to oxidation of thiol to disulfide by PDI. NINSS and TNINSH FONPs were found to be highly efficient in sensing PDI through the AIE-based "fluorescence off/on" mechanism having limits of detection of â¼12.6-17.7 and â¼11.7-16.5 ng/mL, respectively. In vitro cell imaging for NIH3T3 (noncancer) and B16F10 (melanoma) cells with NINSS and TNINSH FONPs displayed excellent diagnosis of eukaryotic cells upon interaction with indigenous PDI. Notably, detection of cancer cells was more sensitive over the noncancerous cells by these FONPs due to overexpression of PDI within cancer cells.
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Dimetil Sulfóxido , Isomerases de Dissulfetos de Proteínas , Camundongos , Animais , Células NIH 3T3 , Naftalimidas , Corantes , Dissulfetos , Oxirredução , Compostos de Sulfidrila , ÁguaRESUMO
Candida auris is a major public health concern due to its high transmission and mortality rates, as well as the emergence of pan-resistant strains. This study aimed to identify an antifungal compound from Sarcochlamys pulcherrima , an ethnomedicinal plant, that can inhibit the growth of C. auris. Methanol and ethyl acetate extracts of the plant were obtained, and high-performance thin-layer chromatography (HPTLC) analysis was conducted to identify the major compounds in the extracts. The major compound detected by HPTLC was subjected to in vitro antifungal activity testing, and its antifungal mechanism was determined. The plant extracts inhibited the growth of both C. auris and Candida albicans. HPTLC analysis revealed the presence of gallic acid in the leaf extract. Furthermore, the in vitro antifungal assay showed that gallic acid inhibited the growth of different C. auris strains. In silico studies indicated that gallic acid can bind to the active sites of carbonic anhydrase (CA) proteins in both C. auris and C. albicans, affecting their catalytic activities. Compounds that target virulent proteins such as CA can aid in the reduction of drug-resistant fungi and the development of novel antifungal compounds with unique modes of action. However, additional in vivo and clinical studies are required to conclusively determine gallic acid's antifungal properties. Gallic acid derivatives may be developed in the future to possess more potent antifungal properties and target various pathogenic fungi.
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BACKGROUND: To compare the performance of conventional, semi-nested and real-time panfungal ITS PCRs for diagnosing fungal keratitis (FK) and develop genus-specific real-time PCR for the most common aetiology of FK. METHODS: This multicentric study includes 232 corneal samples from suspected FK patients from four centres across India between November 2019 through August 2021. A total of 87 corneal buttons were included for the comparison of conventional, semi-nested and real-time ITS PCRs, of which 68 were from confirmed FK patients. Of these 87 samples, 44 (microscopy and culture positive for Aspergillus sp. and/or Fusarium sp.) were used for the standardisation of genus-specific real-time primers/probes. Subsequently, the best method showing highest sensitivity and specificity was validated in 188 samples. RESULTS: On Bayesian comparison, conventional ITS2 PCR showed best performance (sensitivity and specificity of 55.88% and 100%, respectively). Since, real-time ITS2 PCR was also considerably efficient (sensitivity and specificity of 51.47% and 84.21%, respectively) in comparison with the conventional PCR but faster, cost-effective, and less labor-intensive, ITS-2 real-time PCR is a suitable method that can be applied along with culture and microscopy. During validation, real-time PCR with genus-specific primers showed 61.76% and 91.18% sensitivity with specificity of 98.05% and 79.22%, respectively, for Aspergillus sp. and Fusarium sp. Aspergillus probe, Fusarium probe and duplex PCR showed sensitivity of 52.94%, 50% and 54.41% with specificity of 92.86%, 82.47% and 75%, respectively. No cross-reactivity of genus-specific PCRs was observed during standardisation. CONCLUSIONS: ITS-2 real-time PCR can be applied as an adjunct with conventional methods for the diagnosis of FK. The genus-specific duplex real-time PCRs are rapid which reduces the turnaround time (TAT) avoiding the need for sequencing.
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Úlcera da Córnea , Infecções Oculares Fúngicas , Fusarium , Humanos , Fusarium/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Teorema de Bayes , Úlcera da Córnea/microbiologia , Infecções Oculares Fúngicas/diagnóstico , Infecções Oculares Fúngicas/microbiologia , Aspergillus/genética , Sensibilidade e EspecificidadeRESUMO
PURPOSE: Fusarium keratitis possesses significant diagnostic and therapeutic challenges. Medically relevant Fusaria belong to various species complexes and show prominent differences in their antifungal susceptibility profile which may influence the clinical outcome. Rapid diagnostic methods are warranted for precise identification of species complexes for prompt initiation of correct antifungals. The aim of the study was to compare between matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and polymerase chain reaction sequencing for correct species-level identification and to analyze the clinical outcome among different Fusarium species complexes. METHODS: Twenty-nine culture-proven Fusarium keratitis cases were included in this study. A phylogenetic tree was constructed after TEF1α gene sequencing and isolates were subjected to MALDI-TOF MS, followed by database expansion and identification. Clinical outcome and risk association among species complexes were analyzed retrospectively. RESULTS: Maximum likelihood phylogeny categorized 68.9% isolates as Fusarium solani species complex (FSSC), 17.2% as Fusarium dimerum species complex (FDSC), followed by 13.7% as Fusarium fujikuroi species complex (FFSC). With extended database, MALDI-TOF MS could correctly speciate 96.5% (28/29) isolates. Previous antibiotic usage ( P = 0.034) and preoperative antifungal treatment with natamycin, voriconazole, or ketoconazole ( P = 0.025) were significantly higher in the FSSC group. The patients in the FFSC group had a significantly longer duration of symptoms at the time of clinical presentation to the clinic (15 days vs. 5 days, P = 0.030). Among 11 patients with a clinically poor outcome, 9 (31%) had FSSC infection. CONCLUSIONS: Patients infected with the FSSC had more aggressive infection with poor prognosis. MALDI-TOF MS can serve as the best alternative method to conventional molecular identification with reduced turnaround time, which may help the ophthalmologists to consider the appropriate antifungals or early surgical intervention for improved outcome.
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Fusariose , Fusarium , Ceratite , Humanos , Fusarium/genética , Antifúngicos/uso terapêutico , Fusariose/diagnóstico , Fusariose/tratamento farmacológico , Fusariose/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Filogenia , Estudos Retrospectivos , Ceratite/diagnóstico , Ceratite/tratamento farmacológico , Ceratite/microbiologia , Índia/epidemiologia , Atenção à SaúdeRESUMO
We retrospectively reviewed consecutive cases of mucormycosis reported from a tertiary-care center in India to determine the clinical and mycologic characteristics of emerging Rhizopus homothallicus fungus. The objectives were ascertaining the proportion of R. homothallicus infection and the 30-day mortality rate in rhino-orbital mucormycosis attributable to R. homothallicus compared with R. arrhizus. R. homothallicus accounted for 43 (6.8%) of the 631 cases of mucormycosis. R. homothallicus infection was independently associated with better survival (odds ratio [OR] 0.08 [95% CI 0.02-0.36]; p = 0.001) than for R. arrhizus infection (4/41 [9.8%] vs. 104/266 [39.1%]) after adjusting for age, intracranial involvement, and surgery. We also performed antifungal-susceptibility testing, which indicated a low range of MICs for R. homothallicus against the commonly used antifungals (amphotericin B [0.03-16], itraconazole [0.03-16], posaconazole [0.03-8], and isavuconazole [0.03-16]). 18S gene sequencing and amplified length polymorphism analysis revealed distinct clustering of R. homothallicus.
Assuntos
Mucorales , Mucormicose , Humanos , Mucormicose/diagnóstico , Mucormicose/tratamento farmacológico , Mucormicose/microbiologia , Mucorales/genética , Estudos Retrospectivos , Rhizopus/genética , Antifúngicos/farmacologia , Antifúngicos/uso terapêuticoRESUMO
Using infrared spectroscopy and density functional theory (DFT) calculations, we scrutinized an amide (dimethylformamide) as a "model" compound to interpret the interactions of amide 1 with different phenol derivatives (para-chlorophenol (PCP) and para-cresol (CP)) as "model guest molecules". We established the involvement of amide I in vibrational coupling with symmetric and asymmetric C[double bond, length as m-dash]C modes of different phenolic derivatives and how their coupling was dependent upon different guest aromatic phenolic compounds. Interestingly, substitution of phenol perturbed the pattern of vibrational coupling with amide I. The symmetric and asymmetric C[double bond, length as m-dash]C modes of PC were coupled significantly with amide 1. For PCP, the symmetric C[double bond, length as m-dash]C mode coupled significantly, but the asymmetric mode coupled negligibly, with amide I. Here, we reveal the nature of vibrational coupling based on the structure of a guest molecule hydrogen-bonded with amide I. Our conclusions could be valuable for depiction of the unusual dynamics of coupled amide-I modes as well as the dependency of vibrational coupling on altered factors.
RESUMO
PURPOSE: Fungaemia due to yeast is a major cause of morbidity and mortality in critically ill patients. Although, automated blood cultures have improved the time to diagnosis, very few studies have systematically evaluated the utility of blood culture time to positivity (TTP) of fungaemia in the clinical scenario. In this study, we evaluated the TTP for different yeast species to determine its clinical utility. MATERIAL AND METHODS: A prospective study including 244 consecutive patients admitted to the adult (n â= â76) and paediatric (n â= â168) intensive care units (ICUs) was conducted between December 2017 through March 2019. The clinical and demographic characteristics, BACTEC blood culture results and TTP for yeast positive blood cultures were recorded for analysis. RESULTS: A total of 244 patients with 357 episodes of candidaemia were enrolled during the study period. The TTP (mean â± âSD) for all yeast species was 26.8 â± â23.6 âh while it was significantly longer in paediatric than adult patients (30.5 ± 24.7 vs. 25.2 â± â22.9 âh; p â= â<0.0001). Wickerhamomyces anomalus and Cyberlindnera jadinii (previously C. utilis) were exclusively isolated from paediatric population where W. anomalus demonstrated significantly longer TTP than C. jadinii. Among adult cases, C. albicans exhibited significantly longer TTP than C. tropicalis. In paediatric cases, >80% of C. tropicalis and C. utilis flagged positive in blood culture before 24 âh while majority (65.9%) of W. anomalus isolates flagged positive later than 24 âh. Similarly in adult samples, 63% of C. tropicalis isolates beeped positive before 24 âh. CONCLUSION: TTP for yeast may provide insight regarding the responsible yeast species before final identification among critical patients with candidaemia. Larger studies are warranted for evaluating clinical utility of TTP considering other complex factors like yeast burden, generation time, virulence and host factors, which may affect TTP.