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There are approximately 1 million cryptococcal infections per year among HIV+ individuals, resulting in nearly 625,000 deaths. Cryptococcus neoformans and Cryptococcus gattii are the two most common species that cause human cryptococcosis. These two species of Cryptococcus have differences in pathogenicity, diagnosis, and treatment. Cryptococcal infections are usually difficult to identify because of their slow growth in vitro. In addition, the long detection cycle of Cryptococcus in clinical specimens makes the diagnosis of Cryptococcal infections difficult. Here, we used positively charged silver nanoparticles (AgNPs + ) as a substrate to distinguish between C. neoformans and C. gattii in clinical specimens directly via surface-enhanced Raman scattering (SERS) and spectral analysis. The AgNPs + self-assembled on the surface of the fungal cell wall via electrostatic aggregation, leading to enhanced SERS signals that were better than the standard substrate negatively charged silver nanoparticles (AgNPs). The SERS spectra could also be used as a sample database in the multivariate analysis via orthogonal partial least-squares discriminant analysis. This novel SERS detection method can clearly distinguish between the two Cryptococcus species using principal component analysis. The accuracy of the training data and test data was 100% after a tenfold crossover validation.

Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2-3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species.

Objectives: A report on the multicenter evaluation of the Bruker MALDI Biotyper CA System (MBT-CA; Bruker Daltonics, Billerica, MA) for the identification of clinically important bacteria and yeasts. Methods: In total, 4,399 isolates of medically important bacteria and yeasts were assessed in the MBT-CA. These included 2,262 aerobic gram-positive (AGP) bacteria, 792 aerobic gram-negative (AGN) bacteria 530 anaerobic (AnA) bacteria, and 815 yeasts (YSTs). Three processing methods were assesed. Results: Overall, 98.4% (4,329/4,399) of all bacterial and yeast isolates were correctly identified to the genus and species/species complex level, and 95.7% of isolates were identified with a high degree of confidence. The percentage correctly identified and the percentage identified correctly with a high level of confidence, respectively, were as follows: AGP bacteria (98.6%/96.5%), AGN bacteria (98.5%/96.8%), AnA bacteria (98.5%/97.4%), and YSTs (97.8%/87.6%). The extended direct transfer method was only minimally superior to the direct transfer method for bacteria (89.9% vs 86.8%, respectively) but significantly superior for yeast isolates (74.0% vs 48.9%, respectively). Conclusions: The Bruker MALDI Biotyper CA System accurately identifies most clinically important bacteria and yeasts and has optional processing methods to improve isolate characterization.

Fungi of the genus Fusarium are important plant pathogens and contaminants of cereal grains producing different types of mycotoxins. Enniatins are a group of mycotoxins with ionophoric properties frequently detected in North European grains. Within the Fusarium complex responsible for grain infection, Fusarium avenaceum, Fusarium poae and Fusarium tricinctum are the most potential enniatins producers. This study presents the development of two quantitative TaqMan MGB (Minor Groove Binder) assays for the specific quantification of F. avenaceum/F. tricinctum and F. poae esyn1 genotypes, respectively. Two sets of genotype-specific primers/probes were designed on the basis of esyn1 gene homologues encoding multifunctional enzyme enniatin synthetase. The specificity of the assays developed has been tested successfully on 111 Fusarium isolates from different geographical origins. The detection limits for F. avenaceum/F. tricinctum esyn1 genotype and F. poae genotype were 19 and 0.3 pg, respectively. The application of the assays developed on asymptomatic wheat grain samples revealed significant positive correlations between the enniatins levels and the amount of F. avenaceum/F. tricinctum esyn1 genotype (R=0.61) and F. poae esyn1 genotype (R=0.42).

This is a comparative study to isolate the dermatophytes of tinea capitis using the cytobrush and comparing it versus the standard method. A prospective, observational, comparative trial of 178 probable cases of tinea capitis was conducted in two dermatological centers. Each patient underwent mycological tests that included direct exam with KOH and cultures with either of two methods: scraping the scalp to remove hair and cell debris, and the cytobrush. A total of 135 clinically and mycologically proven cases of tinea capitis were included; 119 were non-inflammatory and 16 inflammatory tinea. A total of 131 had a positive direct exam and subsequent primary isolation cultures were obtained in 135 cases. The main dermatophytes isolated were Microsporum canis (68%) and Trichophyton tonsurans (20%). A total of 115/135 (85.1%), were detected with the traditional method, with an average of 11.2 days until positive, while the number detected with the cytobrush was 132/135 (97.7%) with an average of 8.5 days until positive. The chi-square statistical method showed that the cytobrush culture was superior to the standard one with a chi-square of 5.078 (P = 0.025), with a statistically significant difference versus the standard method.