Carbon substrates promotes stress resistance and drug tolerance in clinical isolates of Candida tropicalis.

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.

Clinical spectrum, phenotypic and molecular characterization, and antifungal susceptibility of an emerging human pathogen, Acrophialophora, from India.

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.

A novel indirect ELISA for serodiagnosis of mucormycosis using antigens from Rhizopus arrhizus.

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.

Clinical and Mycologic Characteristics of Emerging Mucormycosis Agent Rhizopus homothallicus.

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.

Naphthalimide-Based AIEgens for Sensing Protein Disulfide Isomerase through Thiol-Disulfide Redox Exchange.

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 ∼12.6-17.7 and ∼11.7-16.5 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.

Metformin-Loaded Hyaluronic Acid-Derived Carbon Dots for Targeted Therapy against Hepatocellular Carcinoma by Glutamine Metabolic Reprogramming.

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 ∼6.5-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 ∼4.9-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.

Naphthalene Diimide-Based Orange Emitting Luminogen: A Fluorometric Probe for Thiol Sensing through the Click Reaction.

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 (∼100-150 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 ∼2.4-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.

Development of single-tube real-time PCR assay for the rapid detection of Aspergillus and Fusarium-The two most common causative agents in fungal keratitis.

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.

Cladosporium halotolerans: Exploring an Unheeded Human Pathogen.

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.

Impact of FKS1 Genotype on Echinocandin In Vitro Susceptibility in Candida auris and In Vivo Response in a Murine Model of Infection.

Echinocandins are frontline antifungal agents in the management of invasive infections due to multidrug resistant Candida auris. The study aimed to evaluate echinocandin resistance in C. auris isolates of multicentric origin, identify the resistance mechanism, and analyze the pharmacodynamic response to caspofungin in a neutropenic mouse model of infection. A total of 199 C. auris isolates originating from 30 centers across India were tested for susceptibility to echinocandins. Isolates with reduced susceptibility were evaluated for FKS1 mutations and in vivo response to caspofungin in a murine model of disseminated candidiasis. In addition, the response to echinocandins was assessed in light of in vitro growth kinetics, chitin content; and transcript levels of chitin synthase and FKS1 genes. We report 10 resistant C. auris isolates with four FKS1 mutations: F635Y (n = 2), F635L (n = 4), S639F (n = 3), and R1354S (n = 1). Of these, F635Y and R1354S exhibited the most profound resistance in mouse model of disseminated infection. S639F and F635L mutations conferred a moderate in vivo resistance, whereas wild-type isolates exhibiting borderline MIC were susceptible in vivo. FKS1 genotype was more accurate predictor of in vivo response than the MIC of the isolates. Isolates with high basal or inducible chitin content exhibited higher in vitro MIC in FKS1 mutant compared to wild type. FKS1 mutations play a major role in clinically relevant echinocandin resistance in C. auris with differential in vivo outcomes. This study could have implications for clinical practice and, therefore, warrants further studies.

Antifungal prescription practices and consumption in a tertiary care hospital of a developing country.

BACKGROUND: Antifungal stewardship is a less explored component of antimicrobial stewardship programmes, especially in developing countries. OBJECTIVE: We aimed to determine antifungal prescription practices in a tertiary centre of a developing country to identify the challenges for antifungal stewardship programmes. METHODS: Four single-day point prevalent surveys were performed in inpatient units and data were collected from medical records. Antifungal use was recorded in terms of consumption, therapeutic strategies and appropriateness. RESULTS: We found a 2.42%-point prevalence of antifungal prescriptions. Antifungal use was higher in children than adults (4.1% vs. 2.03%), medical than surgical units (3.7% vs. 1.24%) and ICUs than general wards (5.8% vs. 1.9%). The highest antifungal use was observed in the haematology-oncology units (29.3%) followed by emergency (16.2%) and gastroenterology units (11.6%). Among 215 prescriptions, amphotericin B was the most commonly prescribed (50.2%) followed by fluconazole (31.6%). The targeted antifungal therapy was practised more commonly (31.5%) than empiric (29.1%), pre-emptive (22.6%) and prophylactic (16.8%) therapy. Amphotericin B was commonly used for pre-emptive (p = .001) and targeted (p = .049) therapy, while fluconazole (p = .001) and voriconazole (p = .011) for prophylaxis. The prescriptions were inappropriate in 25.1% due to the wrong choice of antifungal (44.4%), indication (27.7%) and dosage (24%). The overall mean antifungal consumption was 2.71 DDD/1000 PD and 8.96 DOT/1000 PD. CONCLUSIONS: We report here the low prevalence of antifungal use at a tertiary care centre in a developing country. Though training for antifungal use would be important for antifungal stewardship, the challenge would remain with the affordability of antifungals.

A Selective Medium for Isolation and Detection of Candida auris, an Emerging Pathogen.

Identification of Candida auris is challenging and requires molecular or protein profiling-based approaches, availability of which is limited in many routine diagnostic laboratories, necessitating the development of a cost-effective, rapid, and reliable method of identification. The objective of this study was to develop a selective medium for C. auris identification. Eighteen C. auris and 30 non-C. auris yeasts were used for the standardization of the selective medium. Sodium chloride (10% to 13% concentration) and ferrous sulfate (8 mM to 15 mM) were added to yeast extract-peptone-dextrose (YPD) agar in various combinations followed by incubation at 37°C, 40°C, or 42°C for 2 to 3 days. For validation, 579 yeast isolates and 40 signal-positive Bactec blood culture (BC) broths were used. YPD agar comprising 12.5% NaCl and 9 mM ferrous sulfate incubated at 42°C for 48 h, named Selective Auris Medium (SAM), allowed selective growth of C. auris A total of 95% (127/133) of C. auris isolates tested grew on the standardized media within 48 h, and the remaining 6 isolates grew after 72 h, whereas the growth of 446 non-C. auris yeast isolates was completely inhibited. The specificity and sensitivity of the test medium were both 100% after 72 h of incubation. The positive and negative predictive values were also noted to be 100% after 72 h of incubation. The formulated selective medium can be used for the detection and identification of C. auris The SAM is inexpensive, can easily be prepared, and can be used as an alternative to molecular diagnostic tools in the clinical microbiology laboratory.

Lipase sensing by naphthalene diimide based fluorescent organic nanoparticles: a solvent induced manifestation of self-assembly.

The precise control of supramolecular self-assembly is gaining utmost interest for the demanding applications of manifested nano-architecture across the scientific domain. This study delineates the morphological transformation of naphthalene diimide (NDI) derived amphiphiles with varying water content in dimethyl sulfoxide (DMSO) and the selective sensing of lipase using its aggregation-induced emission (AIE) properties. To this end, NDI-based, benzyl alcohol protected alkyl chain (C1, C5, and C10) linked amphiphilic molecules (NDI-1,2,3) were synthesized. Among the synthesized amphiphiles, benzyl ester linked C5 tailored naphthalene diimide (NDI-2) exhibited AIE with an emission maximum at 490 nm in a DMSO-water binary solvent system from fw = 30% and above water content. The fibrous morphology of NDI-2 at fw = 30% got gradually transformed to spherical aggregated particles along with steady increment in the emission intensity upon increasing the amount of water in DMSO. At fw = 99% water in DMSO, complete transformation to fluorescent organic nanoparticles (FONPs) was observed. Microscopic and spectroscopic techniques demonstrated the solvent driven morphological transformation and the AIE property of NDI-2. Moreover, this AIE of NDI-2 FONPs was employed in the selective turn-off sensing of lipase against many other enzymes including esterase, through hydrolysis of a benzyl ester linkage with a limit of detection 10.0 ± 0.8 µg L-1. The NDI-2 FONP also exhibited its lipase sensing efficiency in vitro using a human serum sample.

Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry: Protocol standardisation, comparison and database expansion for faster and reliable identification of dermatophytes.

BACKGROUND: Accurate and early identification of dermatophytes enables prompt antifungal therapy. However, phenotypic and molecular identification methods are time-consuming. MALDI-TOF MS-based identification is rapid, but an optimum protocol is not available. OBJECTIVES: To develop and validate an optimum protein extraction protocol for the efficient and accurate identification of dermatophytes by MALDI-TOF MS. MATERIALS/METHODS: Trichophyton mentagrophytes complex (n = 4), T. rubrum (n = 4) and Microsporum gypseum (n = 4) were used for the optimisation of protein extraction protocols. Thirteen different methods were evaluated. A total of 125 DNA sequence confirmed clinical isolates of dermatophytes were used to create and expand the existing database. The accuracy of the created database was checked by visual inspection of MALDI spectra, MSP dendrogram and composite correlation index matrix analysis. The protocol was validated further using 234 isolates. RESULT: Among 13 protein extraction methods, six correctly identified dermatophytes but with a low log score (≤1.0). The modified extraction protocol developed provided an elevated log score of 1.6. Significant log score difference was observed between the modified protocol and other existing protocols (T. mentagrophytes complex: 1.6 vs. 0.2-1.0, p < .001; T. rubrum: 1.6 vs. 0.4-1.0, p < .001; M. gypseum:1.6 vs. 0.2-1.0, p < .001). Expansion of the database enabled the identification of all 234 isolates (73.5% with log score ≥2.0 and 26.4% with log scores range: 1.75-1.99). The results were comparable to DNA sequence-based identification. CONCLUSION: MALDI-TOF MS with an updated database and efficient protein extraction protocol developed in this study can identify dermatophytes accurately and also reduce the time for identifying them.

Fungaemia due to rare yeasts in paediatric intensive care units: A prospective study.

BACKGROUND: Considering the emergence of fungaemia due to rare yeasts at our centre, we performed a systematic epidemiologic study on fungaemia due to rare yeast. OBJECTIVES: We undertook the present prospective observational study to explore the epidemiological features and clinical characteristics of fungaemia due to rare yeasts in paediatric ICUs at our centre. METHODS: The successive yeasts isolated from blood at our PICUs during December 2017 through March 2019 were identified by molecular methods. Fungaemia due to yeasts other than C. albicans, C. tropicalis, C. glabrata, C. krusei and C. parapsilosis was categorised as rare yeasts. Antifungal susceptibility testing of the yeast isolates was performed as per clinical and laboratory standards institute (CLSI) guidelines. We also compared different clinical parameters of fungaemia due to common versus rare yeasts, and rare yeasts in neonates versus non-neonates. RESULTS: During the study period, 212 yeast isolates were obtained from 159 patients at PICUs of our hospital, and 127 isolates from 98 patients (61.6%) were categorised as rare yeasts. Neonates acquired fungaemia significantly earlier after ICU admission than non-neonates (median:4 vs 6 days; p = .005). Regarding epidemiology study of rare yeast fungaemia, Wickerhamomyces anomalus (43.8%) and Candida utilis (40.8%) were common isolates; surgical intervention and gastrointestinal disease were significantly associated; overall, azole, echinocandin and amphotericin B resistance was at 9.1%, 1.02% and 1.02%, respectively; overall mortality was 65.3%. CONCLUSIONS: The emergence of rare yeasts especially W. anomalus and C. utilis causing fungaemia in our children demands urgent attention to control the spread.

Rapid and Simple Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantifications of Triazole Antifungals in Human Serum.

PURPOSE: To develop and validate a one-step, rapid and simple reversed-phase high-performance liquid chromatography (HPLC)-based protocol for the simultaneous measurement of voriconazole (VCZ), posaconazole (POSA), itraconazole (ITC) in serum/plasma. METHODS: Calibration standards (CS) and quality control samples were prepared in drug-free serum by spiking with the triazoles at different concentrations. HPLC was performed with C18 column, isocratic mobile phase after extraction with cold acetonitrile. The standardized method was tested in 2693 patients' serum/plasma samples. RESULTS: Linearity of CS for ITC, VCZ and POSA was proportional to the nominal concentration (correlation coefficient > 0.999). Limit of detection (mg/L) for ITC, VCZ and POSA was 0.25, 0.25 and 0.125, respectively. The lower limit of quantification (mg/L) for ITC, VCZ and POSA was 0.5, 0.5 and 0.25, respectively. Precision and accuracy were in acceptable range with 100% average percentage recovery. No interferences from endogenous substances and other antimicrobial compounds were noted. In clinical samples, the therapeutic range achieved for VCZ was 39.9%. Whereas, 61.1% and 44% of samples with ITC and POSA, respectively, were in the sub-therapeutic range. CONCLUSION: We developed a rapid and simple HPLC method to quantify common triazoles in a single chromatographic run allowing simultaneous measurement of different antifungals in a small volume of serum/plasma. Thus, therapeutic drug monitoring requests can be processed in one run without changing the protocol parameters, column or column conditioning thereby improving turnaround time.

Surface-Directed Disparity in Self-Assembled Structures of Small-Peptide l-Glutathione on Gold and Silver Nanoparticles.

Despite the key roles of l-glutathiones (GSHs) inbiology and nano-biotechnology, understanding their labile structures and hydrogen bond interactions with nanoparticles has posed a critical challenge to the scientific community. The structural conformation of GSH as a capping layer on gold nanoparticle (AuNP) and silver nanoparticle (AgNP) surfaces is investigated. In this report, we attempt to explore the material-dependent interaction of GSH with different spherical nanoparticle surfaces by employing Fourier transform infrared (FTIR) spectroscopy. The infrared signal of amide I of GSH is studied as a function of different materials' spherical nanoparticles with comparable size. We revealed the ß-sheet secondary structure of GSH on AgNPs and the random structure on AuNPs even though both the nanoparticles have comparable shapes and sizes and belong to the same group of the periodic table. The GSH is firmly anchored on the gold and silver surface via the thiol of the cys part. However, our experimental data designate a further interaction with the AgNP surface via the carboxylic acid group of the gly- and glu- end of the molecule. It is observed that enhancement of IR absorption of amide I of GSH is pronounced by a factor of 10 on AuNP but, in contrast, on the same-sized AgNP, the suppression is perceived by a factor of 2, even though both are plasmonic materials with respect to free GSH. This study can be used as a point of reference for understanding the structural conformation of the capping layer on nanoparticle surfaces as well as surface enhancement of the IR absorption of amide I. We would like to emphasize that molecular self-assembly on the nanoparticle surfaces is definitely of very broad interest for chemists working in nearly any subdiscipline, spanning from the nanoparticle-based medicine to surface-enhanced spectroscopy to heterogeneous catalysis, etc.

An aero mycological analysis of Mucormycetes in indoor and outdoor environments of northern India.

Mucormycosis is an angio-invasive infection, predominantly acquired by inhalation of sporangiospores from the environment. However, the burden of Mucormycetes sporangiospores in the air is not well studied. We aimed to estimate the burden of Mucormycetes spores in the outdoor and indoor (hospital) environment across different seasons in north India. A total of 380 air samples from outdoor (n = 180) and indoor (n = 200) environment were included in the study. Air samples were suctioned using air sampler (100 l/min) and cultured on Dichloran Rose Bengal Chloramphenicol (DRBC) with benomyl for selective isolation of Mucormycetes. The isolates were identified by phenotypic and genotypic methods. The mean spore count (±SD) of Mucormycetes (cfu/m3) in outdoor samples varied from 0.73 (±0.96) to 8.60 (±5.70) across different seasons. In hospital, the mean spore count varied from 0.68 (±1.07) to 1.12 (±1.07) and 0.88 (±1.01) to 1.72 (±2.17) for air-conditioned wards and non-air-conditioned wards, respectively. Rhizopus arrhizus was the predominant agent isolated from both indoor and outdoor environment followed by Cunninghamella species. We also report a single isolate of the rare mucormycete agent, Apophysomyces variabilis from outdoor environment. The present study highlights the presence of low spore burden of Mucormycetes in outdoor and hospital settings in north India. This study also reports the first isolation of A. variabilis from air samples in the Indian subcontinent.

Mucormycosis due to Apophysomyces species complex- 25 years' experience at a tertiary care hospital in southern India.

Apophysomyces elegans species complex is an important cause of cutaneous mucormycosis in India. However, majority of those cases are reported as case reports only. We desired to analyze our patients with Apophysomyces infection reported over 25 years (1992-2017) to understand the epidemiology, management, and outcome of the disease. During the study period 24 cases were reported, and the majority (95.8%) of them presented with necrotizing fasciitis following accidental/surgical/iatrogenic trauma. One patient presented with continuous ambulatory peritoneal dialysis (CAPD) related peritonitis. Healthcare related Apophysomyces infection was noted in 29.2% patients. In addition to trauma, comorbidities were noted in 37.5% patients (type 2diabetes mellitus-6, chronic alcoholism-2, and chronic kidney disease-1). Of the 24 isolates, 11 isolates starting from year 2014 were identified as Apophysomyces variabilis by molecular methods. Majority (95.8%) of the patients were managed surgically with or without amphotericin B deoxycholate therapy, while one patient was treated with amphotericin B deoxycholate alone. Among 24 patients, seven (29.1%) recovered, six (25%) patients could not afford antifungal management and left the hospital against medical advice, and 11 (45.9%) patients died.The present case series highlights that necrotizing fasciitis caused by A. variabilis is prevalent in India, and the disease may be healthcare related. Although diagnosis is not difficult, awareness among surgeons is still limited about the infection, leading to a delay in sending samples to the mycology laboratory. Apophysomyces infection must be considered in the differential diagnosis in apatient with progressive necrosis of a wound who is not responding to antibacterial therapy.

Branched crack patterns in layers of Laponite® dried under electric fields: Evidence of power-laws and fractal scalin>.

In the present work we report crack patterns formed in aqueous Laponite® gel in a rectangular box, while exposed to a uniform static electric field. The crack pattern shows a very interesting tree-like geometry extending from the positive to the negative electrode. At the positive electrode a large number of cracks appear at first and merge with each other in stages thus forming tree-like fractal structures. These structures are reminiscent of the Bethe lattice or Cayley tree. The "trees" divide the system into peds of varying size, with numerous smaller ones on the positively charged end, gradually increasing in size, and decreasing in number towards the negative end. If the cumulative distribution of the number of peds exceeding a certain area in size, is plotted against that area, a power-law relation is obtained. This implies a scale-invariant fractal character of the pattern. For a given system size, the exponent of the power-law has a nearly constant value for different applied voltages. We present an experimental study demonstrating this behaviour and discuss how it compares with similar distributions of river-basin areas and viscous fingers in a Hele-Shaw cell.