Few studies have systematically standardised and evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of yeasts from bloodstream infections. This is rapidly becoming pertinent for early identification of yeasts and appropriate antifungal therapy. We used 354 yeast strains identified by polymerase chain reaction (PCR) sequencing for standardisation and 367 blind clinical strains for validation of our MALDI-TOF MS protocols. We also evaluated different sample preparation methods and found the on-plate formic acid extraction method as most cost- and time-efficient. The MALDI-TOF assay correctly identified 98.9% of PCR-sequenced yeasts. Novel main spectrum projections (MSP) were developed for Candida auris, C. viswanathii and Kodamaea ohmeri, which were missing from the Bruker MALDI-TOF MS database. Spectral cut-offs computed by receiver operating characteristics (ROC) analysis showed 99.4% to 100% accuracy at a log score of ≥ 1.70 for C. tropicalis, C. parapsilosis, C. pelliculosa, C. orthopsilosis, C. albicans, C. rugosa, C. guilliermondii, C. lipolytica, C. metapsilosis, C. nivariensis. The differences in the species-specific scores of our standardisation and blind validation strains were not statistically significant, implying the optimal performance of our test protocol. The MSPs of the three new species also were validated. We conclude that MALDI-TOF MS is a rapid, accurate and reliable tool for identification of bloodstream yeasts. With proper standardisation, validation and regular database expansion, its efficiency can be further enhanced.

To assess the prevalence of fungal keratitis, we conducted a retrospective study over 7 years (2005 through 2011) at a tertiary care center in North India. Effort has been made to analyze the disease burden, spectrum of agents and treatment history. The findings were compared with an earlier study at the same center for any change in the epidemiology of the disease. Microbiology records were screened at the Postgraduate Institute of Medical Education and Research, Chandigarh, India, to identify fungal keratitis cases, and available clinical records of those cases were analyzed. Of 2459 clinically suspected fungal keratitis cases, 765 (31 %) cases were direct microscopy confirmed. Of these microscopy-confirmed cases, fungi were isolated in 393 (51.4 %), with Aspergillus spp. ranked top (n = 187, 47.6 %), followed by melanized fungi (n = 86, 21.9 %) and Fusarium spp. (n = 64, 16 %). A male predominance of 78.7 % was noted with a peak in the incidence of fungal keratitis during post-monsoon season (September to November). A delay in diagnosis was significantly associated (p < 0.001) with keratitis cases due to melanized fungi. In comparison with an earlier study, higher isolation of melanized fungi was noted with a widening of the spectrum of agents identified. Thus, fungal keratitis due to Aspergillus spp. remains a serious ocular illness among the active male population in North India with relative rise of keratitis due to melanized fungi. The spectrum of agents causing fungal keratitis has broadened with many rare fungi that are implicated.

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A mussel mimetic transdermal patch was prepared using bacterial cellulose (BC), a green resource derived from Glucanoacetobacter xylinus (MTCC7795). To impart the mussel mimetic property, dopamine (DOPA), a catechol-containing compound, was used to modify the isolated BC via an amidation reaction between the carboxylated BC and DOPA, and the end product was successively characterized by 1H NMR and FTIR analysis. The free hydroxyl group of the DOPA moiety of DOPA-modified BC (BC-DOPA) was utilized to prepare BC-DOPA/rGO/Ag NPs, a composite film incorporating reduced graphene oxide/silver nanoparticles (rGO/Ag NPs). The antimicrobial action of the prepared film was determined against both Gram-positive (Staphylococcus aureus and Lysinibacillus fusiformis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. The bactericidal property of the composite film was determined using the zone of inhibition (ZOI) method and live–dead assay (DAPI–PI analysis). The morphological transformation of bacteria upon the application of the composite film was observed through SEM analysis. The cell compatibility of the composite film over the NIH 3T3 fibroblast cell line was assessed through an XTT assay. The in vitro wound-healing assays over the NIH 3T3 cell line and A549 human lung epithelial cell line reveal that the presence of rGO and Ag NPs in the composite film accelerates the wound-healing process.

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Matrix-assisted laser desorption ionization-time of flight mass spectrometry can be used for routine identification of clinically important molds.

With the changing epidemiology and emergence of antifungal resistance among Candida species, rapid antifungal susceptibility testing (AFST) is crucial for optimization of antifungal therapy. This study was conducted to standardize a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI -TOF MS) based AFST method (ms-AFST) for susceptibility of Candida tropicalis isolates. Clinical isolates of C. tropicalis were confirmed for fluconazole resistance by the CLSI (M27-A3) method. The incubation period and drug concentration were optimized to determine the minimal profile change concentration (MPCC) by MALDI-TOF MS. The data were analyzed first by direct visual observation of the spectra followed by composite correlation index (CCI) matrix analysis, virtual gel analysis, and cluster analysis for confirmation. Finally, the correlation between minimum inhibitory concentrations (MICs) and MPCCs was evaluated. A total of 15 fluconazole resistant (MICs ranging from 16 to 128 μg/ml) and 19 fluconazole susceptible C. tropicalis isolates (MIC ≤1 μg/ml) were included in this study. All C. tropicalis isolates had significant spectral changes after 4h incubation with fluconazole. Of 34 isolates, MPCCs and MICs were equivalent for 16 isolates, and the MPCC was one dilution lower than the respective MIC in the remaining 18 isolates. This finding was further supported by visual analysis, CCI matrix analysis, virtual gel and principal component analysis dendrogram analysis. The correlation between MPCC and MIC was significant (P < .05). Therefore, a MALDI-TOF MS based AFST assay may be used as a rapid screening technique for fluconazole resistance in C. tropicalis.

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