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.

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.

Evaluation of environmental Mucorales contamination in and around the residence of COVID-19-associated mucormycosis patients.

Introduction: Recently, India witnessed an unprecedented surge of coronavirus disease 2019 (COVID-19)-associated mucormycosis (CAM) cases. In addition to patient management issues, environmental Mucorales contamination possibly contributed to the outbreak. A recent study evaluated environment contamination by Mucorales in the hospital setting. However, a considerable number of CAM patients were never admitted to a hospital before the development of the disease. The present study, therefore, planned to evaluate Mucorales contamination of patients' residences. Methods: The residential environment of 25 patients with CAM living in north India was surveyed. Air samples were collected from indoor and immediate outdoor vicinity of the patients' residence and cultured on Dichloran Rose-Bengal Chloramphenicol (DRBC) agar with benomyl for selective isolation of Mucorales. Surface swab samples were also collected from the air coolers fitted in those residences and cultured on DRBC agar. The isolates were identified by phenotypic and genotypic methods. Amplified fragment length polymorphism (AFLP) was employed to evaluate the genetic relatedness of the environmental and patients' clinical isolates. Results: The median spore count (mean ± SD, cfu/m3) of Mucorales in the air of patients' bedrooms was significantly higher than in the air in other rooms in those residences (3.55 versus 1.5, p = 0.003) or the air collected directly from the front of the air cooler (p < 0.0001). The Mucorales spore count in the environment did not correlate with either ventilation of the room or hygiene level of the patients' residences. Rhizopus arrhizus was isolated from the environment of all patients' residences (n = 25); other Mucorales species isolated were Cunninghamella bertholletiae (n = 14), Rhizopus microsporus (n = 6), Rhizopus delemar (n = 6), Syncephalastrum racemosum (n = 1), Lichtheimia corymbifera (n = 1), and Mucor racemosus (n = 1). Genetic relatedness was observed between 11 environmental isolates from the patients' bedrooms and respective clinical isolates from patients. Discussion: The study supported the view that the patients might have acquired Mucorales from the home environment during the post-COVID-19 convalescence period. Universal masking at home during patients' convalescence period and environmental decontamination could minimize exposure in those susceptible patients.

Mechanisms of azole antifungal resistance in clinical isolates of Candida tropicalis.

This study was designed to understand the molecular mechanisms of azole resistance in Candida tropicalis using genetic and bioinformatics approaches. Thirty-two azole-resistant and 10 azole-susceptible (S) clinical isolates of C. tropicalis were subjected to mutation analysis of the azole target genes including ERG11. Inducible expression analysis of 17 other genes potentially associated with azole resistance was also evaluated. Homology modeling and molecular docking analysis were performed to study the effect of amino acid alterations in mediating azole resistance. Of the 32 resistant isolates, 12 (37.5%) showed A395T and C461T mutations in the ERG11 gene. The mean overexpression of CDR1, CDR3, TAC1, ERG1, ERG2, ERG3, ERG11, UPC2, and MKC1 in resistant isolates without mutation (R-WTM) was significantly higher (p<0.05) than those with mutation (R-WM) and the sensitive isolates (3.2-11 vs. 0.2-2.5 and 0.3-2.2 folds, respectively). Although the R-WTM and R-WM had higher (p<0.05) CDR2 and MRR1 expression compared to S isolates, noticeable variation was not seen among the other genes. Protein homology modelling and molecular docking revealed that the mutations in the ERG11 gene were responsible for structural alteration and low binding efficiency between ERG11p and ligands. Isolates with ERG11 mutations also presented A220C in ERG1 and together T503C, G751A mutations in UPC2. Nonsynonymous mutations in the ERG11 gene and coordinated overexpression of various genes including different transporters, ergosterol biosynthesis pathway, transcription factors, and stress-responsive genes are associated with azole resistance in clinical isolates of C. tropicalis.

Clinical and mycological profile of fungal keratitis from North and North-East India.

Purpose: To study the clinical presentation, mycological profile, and risk factors of fungal keratitis (FK) cases presenting at two tertiary-care centers, one each at North (Chandigarh) and Northeast (Assam) India, and to compare the spectrum of fungi recovered from the clinical and environmental samples at both locations. Methods: All patients with suspected FK were enrolled from both the centers between January 2018 and December 2019. Corneal samples were collected and processed as per standard laboratory protocols. Demographic details and clinical and mycological profiles were noted in all patients. Environmental sampling from the soil, air, and the vegetative matter was performed from both locations and neighboring districts. Results: Of the 475 suspected cases, 337 (71%) were diagnosed as FK (median age: 50 years; 77.2% males). The presence of diabetes, hypertension, blurred vision, and corneal discoloration was significantly higher in patients with FK compared to those without FK. Aspergillus sp. (52.1%) and Fusarium sp. (47.61%) were the predominant etiological agents isolated from cases in North and Northeast India, respectively. FK due to melanized fungi was associated with diabetes, trauma with animal tail, and corneal discoloration. A similar spectrum of fungi was seen in environmental and clinical samples in both the regions. Conclusion: The difference in etiological agents of FK and environmental fungal isolates in North and Northeast India highlights the need to identify the ecological niche of potential fungal pathogens. Prospective, multicenter studies, systematic environmental sampling, and the evaluation of the differences in causative agents and clinical presentation of FK from different parts of the country can substantially improve our understanding of its region-specific clinico-epidemiological profile.

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.

Expression and Purification along with Evaluation of Serological Response and Diagnostic Potential of Recombinant Sap2 Protein from C. parapsilosis for Use in Systemic Candidiasis.

Systemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Although C. albicans is a predominant species causing systemic candidiasis, infections caused by non-albicans Candida (NAC) species are increasingly becoming more prevalent globally along with the emergence of drug resistance. The diagnosis of systemic candidiasis is difficult due to the absence of significant clinical symptoms in patients. We investigated the diagnostic potential of recombinant secreted aspartyl proteinase 2 (rSap2) from C. parapsilosis for the detection of Candida infection. The rSap2 protein was successfully cloned, expressed and purified using Ni-NTA chromatography under denaturing conditions using an E. coli-based prokaryotic expression system, and refolded using a multi-step dialysis procedure. Structural analysis by CD and FTIR spectroscopy revealed the refolded protein to be in its near native conformation. Immunogenicity analysis demonstrated the rSap2 protein to be highly immunogenic as evident from significantly high titers of Sap2-specific antibodies in antigen immunized Balb/c mice, compared to sham-immunized controls. The diagnostic potential of rSap2 protein was evaluated using immunoblotting and ELISA assays using proven candidiasis patient serum and controls. Immunoblotting results indicate that reactivity to rSap2 was specific to candidiasis patient sera with no cross reactivity observed in healthy controls. Increased levels of anti-Sap2-specific Ig, IgG and IgM antibodies were observed in candidiasis patients compared to controls and was similar in sensitivity obtained when whole Candida was used as coating antigen. In summary, the rSap2 protein from C. parapsilosis has the potential to be used in the diagnosis of systemic candidiasis, providing a rapid, convenient, accurate and cost-effective strategy.

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.

Rapid detection of ERG11 polymorphism associated azole resistance in Candida tropicalis.

Increasing reports of azole resistance in Candida tropicalis, highlight the development of rapid resistance detection techniques. Nonsynonymous mutations in the lanosterol C14 alpha-demethylase (ERG11) gene is one of the predominant mechanisms of azole resistance in C. tropicalis. We evaluated the tetra primer-amplification refractory mutation system-PCR (T-ARMS-PCR), restriction site mutation (RSM), and high-resolution melt (HRM) analysis methods for rapid resistance detection based on ERG11 polymorphism in C. tropicalis. Twelve azole-resistant and 19 susceptible isolates of C. tropicalis were included. DNA sequencing of the isolates was performed to check the ERG11 polymorphism status among resistant and susceptible isolates. Three approaches T-ARMS-PCR, RSM, and HRM were evaluated and validated for the rapid detection of ERG11 mutation. The fluconazole MICs for the 12 resistant and 19 susceptible isolates were 32-256 mg/L and 0.5-1 mg/L, respectively. The resistant isolates showed A339T and C461T mutations in the ERG11 gene. The T-ARMS-PCR and RSM approaches discriminated all the resistant and susceptible isolates, whereas HRM analysis differentiated all except one susceptible isolate. The sensitivity, specificity, analytical sensitivity, time, and cost of analysis suggests that these three methods can be utilized for the rapid detection of ERG11 mutations in C. tropicalis. Additionally, an excellent concordance with DNA sequencing was noted for all three methods. The rapid, sensitive, and inexpensive T-ARMS-PCR, RSM, and HRM approaches are suitable for the detection of azole resistance based on ERG11 polymorphism in C. tropicalis and can be implemented in clinical setups for batter patient management.

Fatal cerebral phaeohyphomycosis caused by Cladophialophora bantiana mimicking tuberculous brain abscess.

INTRODUCTION: Cladophialophora bantiana, a neurotropic phaeoid fungus, is the primary agent of cerebral phaeohyphomycosis. The disease more commonly affects immunocompetent males and is associated with a high mortality rate. CASE REPORT: We report a case of brain abscess caused by Cladophialophora bantiana in a 50-year-old immunocompetent male who presented with headache for two months, weakness of both lower limbs for 15 days, and altered sensorium and aphasia for one day. Contrast-enhanced MRI of the brain showed multiple coalescent abscesses in the right basal ganglia and corpus callosum. Based on clinical and radiological suspicion of tuberculoma, treatment with antitubercular drugs was initiated. A month after discharge, the patient was re-admitted with history of loss of consciousness, altered sensorium, respiratory distress and aphasia. Brain CECT revealed multiple ring-enhancing lesions in the right basal ganglia with mass effect and a leftward midline shift. The patient underwent craniotomy and evacuation of abscess. Direct microscopy of pus aspirated from the lesions showed pigmented septate fungal hyphae, which was identified as C. bantiana in fungal culture. The patient was administered intravenous liposomal amphotericin B and voriconazole. However, he died due to multiple organ failure on day 19 after surgery. CONCLUSIONS: Fungal etiology should be considered in the differential diagnosis of intracranial space occupying lesions, regardless of the host immune status. An early diagnosis, together with aggressive medical and neurosurgical interventions are imperative for improving the survival in such patients.

Phenotypic and molecular characterisation of Sporothrix globosa of diverse origin from India.

Sporotrichosis is one of the neglected tropical diseases causing subcutaneous chronic granulomatous lesion by thermally dimorphic fungi belonging to Sporothrix species. Sporothrix brasiliensis, Sporothrix mexicana and Sporothrix globosa are the common pathogenic species. In Asian countries, S. globosa constitutes nearly 99.3% of all Sporothrix species. We studied 63 cases of sporotrichosis of geographically diverse origin from India and Sporothrix isolates were characterised for its growth in different media, temperatures, ability to assimilate sugars and antifungal susceptibility profile. Molecular characterization was performed by sequencing of the calmodulin (CAL), beta tubulin (BT) and translational elongation factor 1-alpha (TEF-1α) and typing by fluorescent amplified fragment length polymorphism (FAFLP). In patients who presented with fixed (49.2%), lymphocutaneous lesions (23.8%), in 26.9% the details were not known, none had systemic dissemination. All the isolates tested were Sporothrix globosa and that could grow up to 35 °C and unable to grow at and beyond 37 °C. The assimilation of sucrose, ribitol and raffinose helps in identifying S. globosa. Sequences of CAL or BT or TEF-1α can differentiate S. globosa from other species in the complex. FAFLP results exhibited low genetic diversity. No correlation was noted between genotypes and clinical presentation, or geographic distribution. Itraconazole, terbinafine and posaconazole showed good in vitro antifungal activity against S. globosa whereas fluconazole and micafungin had no activity. S. globosa of Indian origin is relatively less pathogenic than other pathogenic Sporothrix species as it does not cause systemic dissemination and in the diagnostic laboratory, incubation of the cultures below 37 °C is essential for effective isolation.

Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues.

Introduction. Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species levelGap Statement Available protocols for the molecular identification of fungi from formalin-fixed and paraffin-embedded (FFPE) tissues have limitations in terms of extraction and target selection, and standardisation.Aim. Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues.Methodology. A total of 63 FFPE tissues from histopathology proven IFI cases were used to standardize the DNA extraction (commercial QIAamp kit-based extraction and conventional phenol-chloroform-isoamyl alcohol [PCI] method) and sequence-based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8S-ITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Semi-nested PCR targeting Mucorales-specific 18S rDNA region was performed in tissues having aseptate hyphae. The optimized ITS1-PCR protocol was evaluated in 119 FFPE tissues containing septate hyphae or yeast, and Mucorales-specific semi-nested PCR in 126 FFPE tissues containing aseptate hyphae.Results. The DNA yield by conventional PCI method was significantly higher (P<0.0001) than commercial kit, though the quality of DNA was similar by both protocols. The test accuracy was best while using ITS1 (61.9 %) as the target compared to 7.9, 29.9 and 22.2 % on targeting ITS1-5.8S-ITS2, ITS2, the D1/D2 region of 28S, respectively. The test accuracies of ITS1-PCR in tissues containing septate hyphae, aseptate hyphae and yeasts were 75.5, 18.7 and 100 %, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50 µm) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucorales-specific semi-nested PCR in 86 (68.3 %) tissues.Conclusion. Conventional PCI-based DNA extraction from thick tissue (50 µm) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1-PCR for septate fungi and yeast, and semi-nested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.

Hydrothermal synthesis of gelatin quantum dots for high-performance biological imaging applications.

In the present study, we are reporting a one-pot synthesis of gelatin quantum dots (GeQDs) by the hydrothermal process. The synthesized GeQDs were characterized by fourier transform infrared spectroscopy, nuclear magnetic resonance, ultraviolet-visible and photoluminescence spectroscopic techniques, and also by using high-resolution transmission electron microscopy. The GeQDs showed a high level of photoluminescence quantum yield (PLQY) with significantly higher stability for up to 6 months and presented similar fluorescent intensity as the initial PLQY without any precipitation and aggregation at ambient condition. The cell imaging ability of synthesized GeQDs was examined using cells belonging to diverse clinical backgrounds like bacterial cells including Escherichia coli and Staphylococcus aureus, yeast cells including Candida albicans, C. krusei, C. parapsilosis, and C. tropicalis, mycelial fungi including Aspergillus flavus and A. fumigatus cells, cancer cell lines A549, HEK293 and L929. The results demonstrated that the GeQDs illuminates the cells and can be utilized as potential cell labeling non-toxic biomarkers. In conclusion, it can be said that the gelatin stabilized QDs are a promising candidate for stable and long-term fluorescent imaging of different types of cells.

Dynamics of in vitro development of azole resistance in Candida tropicalis.

OBJECTIVES: Increasing incidence of azole resistance in Candida tropicalis, especially to fluconazole, has been seen in Asian countries including India. Limited knowledge is available on the molecular mechanisms associated with the development of azole resistance in C. tropicalis. The present study examined the dynamics of in vitro azole resistance in C. tropicalis after prolonged treatment with fluconazole. METHODS: Nine fluconazole-susceptible isolates of C. tropicalis were used in this study. Fluconazole resistance was induced experimentally in C. tropicalis isolates. The stability of induced resistance and cross-resistance to other azoles was examined. The molecular mechanisms of azole resistance were assessed by measuring the expression and mutation analysis of different genes. RESULTS: Varying degrees of resistance [five with minimum inhibitory concentrations (MICs) ≤32 mg/L and four with MICs ≥128 mg/L] were noticed, and the resistance was developed in 3 months. Of the nine resistant isolates, four induced resistant isolates with MICs ≥128 mg/L presented temporal resistance stability up to 10 subcultures. These four isolates presented cross-resistance to other azoles and also an inducible overexpression of transporters (CDR1, CDR2, CDR3 and MDR1), ergosterol biosynthesis pathway genes (ERG1, ERG2, ERG3 and ERG11), transcription factors (TAC1 and UPC2) and stress-responsive genes (HSP90 and MKC1) was noticed. No mutations were seen in any of the four genes (ERG1, ERG3, ERG11 and UPC2) tested. CONCLUSIONS: Candida tropicalis isolates adapt themselves in the presence of continuous drug exposure and switch back to being susceptible in the absence of the drug. The acquisition of resistance in C. tropicalis is mediated by the overexpression of different resistance-related genes without any molecular alterations.

Selection and evaluation of appropriate reference genes for RT-qPCR based expression analysis in Candida tropicalis following azole treatment.

Candida tropicalis arises as one of the predominant non-Candida albicans Candida (NCAC) species causing invasive candidiasis in Asian countries. A rise in reports of C. tropicalis with a parallel increase in fluconazole resistance has also been observed. The genes and underlying pathways associated with azole antifungal resistance in C. tropicalis is still not properly understood. The RT-qPCR is the most promising approach for expression analysis of target genes to understand the mechanisms of resistance. The reliability and reproducibility of this technique depend on the selection of suitable reference genes for the normalization in expression study. The present study investigated the expression stability levels of ten genes including ACT1, EF1, GAPDH, PGK1, RDN5.8, RDN18, RDN28, SDHA, TUB1, and UBC13 for their suitability in fluconazole treated/untreated C. tropicalis. The stability levels of these genes were examined by the ∆∆CT, ΔCT, Pfaffl methods and five independent software including hkgFinder, geNorm, NormFinder, BestKeeper, and RefFinder software. We report, the EF1 and ACT1 were the most stable reference genes for normalization and can be used for the gene expression analysis in C. tropicalis. To the best of our knowledge, our study is the first to select and validate the reference genes in C. tropicalis for RT-qPCR based expression analysis.

Prolonged Outbreak of Candida krusei Candidemia in Paediatric Ward of Tertiary Care Hospital.

BACKGROUND: A sudden rise of Candida krusei candidemia cases was noticed in our hospital within 1 year with maximum cases from paediatric unit. The present study reports the results of epidemiological investigation of possible outbreak of candidemia by C. krusei in paediatric unit at our tertiary care centre. METHODS: Clinical characteristics and risk factors associated with C. krusei candidemia were evaluated. Yeast identification and antifungal susceptibility testing was performed according to standard protocol. To find the potential source of C. krusei in hospital environment and hand colonization, swabs were collected from different fomites (n = 40) and hand washings from 24 health care workers (HCW), respectively. Infection control and prevention practices were intensified following the recognition of outbreak. Genetic typing was done by fluorescent amplified fragment length polymorphism (FAFLP) technique. Case-control comparison was performed with C. tropicalis and C. pelliculosa cases. RESULTS: Candida krusei fungaemia significantly affected paediatric group (82/186, 44%) as compared to adults (14/130, 10.8%; p < 0.001). Among paediatric group, maximum isolation was reported from neonatal unit of paediatric emergency (NUPE). C. krusei was isolated from hands of one HCW and washbasin in NUPE. FAFLP revealed clonality between blood and environmental isolates indicating cross-transmission of C. krusei. Gastrointestinal disease (p = 0.018), previous antibiotics (p = 0.021) especially to carbapenems (p = 0.039), was significant among C. krusei candidemia cases compared to C. pelliculosa cases. CONCLUSION: We report the largest outbreak of C. krusei candidemia in paediatric unit within 1 year with isolation of related strains from environment and hands of HCW. Routine screening of hand hygiene practices revealed non-compliance to standard practices leading to the increase in C. krusei candidemia cases.

Probing the Griffiths like phase, unconventional dual glassy states, giant exchange bias effects and its correlation with its electronic structure in Pr2-x Sr x CoMnO6.

Crystal, electronic structure, dc and ac magnetization properties of the hole substituted (Sr2+) and partially B-site disordered double perovskite Pr2-x Sr x CoMnO6 system have been investigated. The XRD pattern analysis showed a systematic decrease in the lattice parameters owing to the enhanced oxidation states of the Co/Mn ions. The electronic structure study by XPS measurements suggested the presence of mixed valence states of the B-site ions (Co2+ /Co3+ and Mn3+ /Mn4+) with significant enhancement of the average oxidation states due to hole doping. The mere absence of electronic states near the Fermi level in the valence band (VB) spectra for both pure (x = 0.0) and Sr doped (x = 0.5) systems indicated the insulating nature of the samples. Sr substitution is observed to increase the spectral weight near the Fermi level suggesting for an enhanced conductivity of the hole doped system. The dc magnetization data divulged a Griffiths like phase above the long-range ordering temperature. A typical re-entrant spin glass like phase driven by the inherent anti-site disorder (ASD) has been recognized by ac susceptibility study for both the pure and doped systems. Most interestingly, the emergence of a new cluster glass like phase (immediately below the magnetic ordering temperature and above the spin-glass transition temperature) solely driven by the Sr substitution has been unravelled by ac magnetization dynamics study. Observation of these dual glassy states in a single system is scarce and hence placed the present system amongst the rare materials. The isothermal magnetization measurements further probed the exhibition of the giant exchange bias effect originated from the interfacial exchange interactions due to existence of low temperature antiferromagnetic clusters embedded in the glassy matrix.

Stable isotope labelling: an approach for MALDI-TOF MS-based rapid detection of fluconazole resistance in Candida tropicalis.

BACKGROUND: With the rise of fluconazole resistance in Candida tropicalis in Asian countries, the rapid detection of resistance is required to optimize therapy. OBJECTIVES: We evaluated a stable isotope labelling by/with amino acids in cell culture (SILAC) approach for rapid detection of fluconazole resistance in C. tropicalis by MALDI-TOF MS. METHODS: Twenty-six fluconazole-resistant and 19 -susceptible C. tropicalis isolates were used. Isolates were grown in media containing normal lysine (NL), isotopically labelled ('heavy') lysine (HL) and fluconazole (FLC) with labelled lysine (HL + FLC). MALDI-TOF MS was performed, acquired spectra were visually compared and composite correlation index (CCI) values were calculated. The results were analysed by CCI matrix, virtual gel and principal component analysis (PCA). RESULTS: The MICs of fluconazole in 26 resistant and 19 susceptible isolates were 16-256 mg/L and 0.5-1 mg/L, respectively. The m/z values of 15 peaks specific for media containing NL and their corresponding peaks specific for media with HL were detected to differentiate resistant and susceptible isolates. The CCI cut-off values for susceptible and resistant isolates were significant (P < 0.05). The CCI matrix, virtual gel and PCA dendrogram confirmed the results. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of this method for detection of fluconazole resistance were 92.31%, 100%, 100%, 90.48% and 95.56%, respectively. CONCLUSIONS: SILAC, a promising approach for rapid resistance detection in C. tropicalis using MALDI-TOF MS, may be used in the routine laboratory in the near future.

MALDI-TOF MS-Based Identification of Melanized Fungi is Faster and Reliable After the Expansion of In-House Database.

PURPOSE: Invasive fungal infections caused by melanized fungi are a growing concern. Rapid and reliable identification plays an important role in optimizing therapy. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based identification has emerged as a faster and more accurate diagnostic technique. However, lack of a protein extraction protocol and limited database restricts the identification of melanized fungi by MALDI-TOF MS. The study is designed to standardize protein extraction protocol, to enrich the existing, and to create an in-house database for the rapid identification of melanized fungi. EXPERIMENTAL DESIGN: In this study, 59 sequence-confirmed, melanized fungi were used to expand and to create an in-house database using a modified protein extraction protocol. A total of 117 clinical isolates are further used to validate the created database. RESULT: Using existing Bruker database, only 29(24.8%) out of 117 moulds could be identified. However, all the isolates are identified accurately by supplementing the Bruker database with the created in-house database. MALDI-TOF MS takes significantly lesser time for identification compared to DNA sequencing. CONCLUSION AND CLINICAL RELEVANCE: An expanded database with modified protein extraction protocol can reduce significant time to identify melanized fungi by MALDI-TOF MS.