White Piedra: Clinical, Mycological, and Therapeutic Experience of Fourteen Cases.

BACKGROUND: White piedra (WP) is an asymptomatic superficial mycosis that affects the hair stems, forming whitish nodules caused by various species of the genus Trichosporon. OBJECTIVE: To present a case series of WP of the head, its epidemiological data, as well as clinical, mycological, and therapeutic experience. METHODS: We conducted a 12-year retrospective and observational study of WP cases tested by dermoscopy, mycological study, and the identification of species through morphology, biochemistry, and proteomics (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). The treatment was based on ketoco-nazole shampoo as well as keratolytics. RESULTS: We included 14 cases of WP, all located in the head and 1 case with both head and scrotum affected. Nine cases (64.3%) presented in children aged < 15 years. The majority of the cases (13/14, 92.8%) were women. Two cases were associated with hyperkeratosis and intertrigo. Most patients had long hair and excessive moisture. In all cases hair nodules were observed and Trichosporon inkin (11/14, 78.6%) was usually isolated. Eleven cases (78.6%) were cured by administering 2% ketoconazole shampoo. CONCLUSION: WP was observed in school-age girls. The diagnosis was based on the observation of hair nodules and its main etiologic agent was T. inkin, with good response to treatment in most cases.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric identification and antifungal susceptibility analysis of Candida species isolated from patients with invasive yeast infections in five university hospitals.

In this multicenter study, we compared the performance of the Bruker Biotyper MS system and VITEK 2 YST systems for invasive yeast identification, investigated the distribution of isolated species, and evaluated the antifungal susceptibility profiles of Candida albicans, Candida parapsilosis, and Candida tropicalis. In cases of discrepant results lack of identification with either method, molecular identification techniques were employed. We tested 216 clinical isolates, and concordance between the two methods was observed for 192/216 isolates (88.9%). For five unidentified strains (2.3%), an internal transcribed spacer (ITS) sequencing approach was used. In brief, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) provided short turnaround times and more reliable results than those of Vitek 2 YST. In Wuhan, C. albicans, C. parapsilosis, Candida glabrata, and C. tropicalis were the most common pathogens (93.0%) in patients with candidemia. Cryptococcus neoformans was mainly detected in cerebrospinal fluid samples (88.9%). Trichosporon asahii were all isolated from drainage fluids in the Surgery. Candida albicans was clearly susceptible to azoles, while C. parapsilosis and C. tropicalis displayed differences in susceptibility to azoles. Our findings provide a basis for the practical application of MALDI-ToF MS for identification and for the use of ATB FUNGUS 3 to characterize the susceptibility of Candida spp., thereby providing significant data for therapeutic decisions.

Evaluation of MALDI-TOF mass spectrometry (VITEK-MS) compared to the ANC card (VITEK 2) for the identification of clinically significant anaerobes / Avaliação da espectrometria de massas MALDI-TOF (VITEK-MS) diante do cartão ANC (VITEK 2) na identificação de anaeróbios clinicamente significantes

ABSTRACT Introduction: The identification of anaerobic bacteria by conventional methods employed in clinical laboratories requires a lot of work and a long response time [turnaround time (TAT)]. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an accurate, rapid and inexpensive technique with promising results for bacterial identification. Objective: To evaluate the MALDI-TOF mass spectrometry (VITEK-MS, bioMérieux, France) compared to the ANC card (VITEK 2, bioMérieux, France) for the identification of anaerobes, and also veriying the cost variation between both methodologies. Methods: 421 anaerobes were concomitantly identified by ANC (VITEK 2) and MALDI-TOF (VITEK MS). The conflicting results or those presenting low differentiation of the species were subjected to the 16S ribosomal ribonucleic acid (rRNA) sequencing. Results: Thirty-five strains were not identified by ANC (VITEK 2), and only one isolate was not identified by MALDI-TOF (VITEK MS). From the 386 anaerobes identified by the two methodologies, 97% agreement was observed on the identification of genus and species between the methodologies. Thirteen (3%) isolates were submitted to 16S sequencing. The agreement observed was 70% using ANC (VITEK 2) using 92% by MALDI-TOF (VITEK MS). Conclusion: Both methodologies showed an excellent performance for the identification of the strains tested with great differences in relation to cost-benefit. MALDI-TOF MS allowed 35 additional identifications and a saving of BRL$ 7,786 with the release of culture positive result five days ahead of the ANC (VITEK 2). TAT reduction may contribute to a successful clinical resolution.

RESUMO Introdução: A identificação das bactérias anaeróbias por métodos convencionais empregados nos laboratórios clínicos demanda muito trabalho e um longo tempo de resposta (TAT). A espectrometria de massa por ionização e dessorção a laser assistida por matriz (MALDI-TOF MS) é uma técnica precisa, rápida e barata, com resultados promissores para a identificação bacteriana. Objetivo: Avaliar a espectrometria de massas MALDI-TOF (VITEK MS, bioMérieux, France) diante do cartão ANC (VITEK 2, bioMérieux, France) para a identificação de anaeróbios, bem como verificar a variação de custos entre as metodologias. Métodos: Foram identificados 421 anaeróbios concomitantemente pelo ANC (VITEK 2) e pelo MALDI-TOF (VITEK MS). Os resultados discordantes ou que apresentaram baixa discriminação das espécies foram submetidos ao sequenciamento do 16S do ácido ribonucleico ribossonal (rRNA). Resultados: Trinta e cinco cepas não foram identificadas pelo ANC (VITEK 2), e somente um isolado ficou sem identificação pelo MALDI-TOF (VITEK MS). Dos 386 anaeróbios identificados pelas duas metodologias, a concordância na identificação de gênero e espécie foi observada em 97%. Treze (3%) isolados foram submetidos ao sequenciamento do 16S; a concordância observada foi de 70% com o ANC (VITEK 2) e 92% com MALDI-TOF (VITEK MS). Conclusão: Ambas as metodologias demonstraram ótimo desempenho para identificação das cepas testadas com grandes diferenças em relação ao custo-benefício. O MALDI-TOF MS permitiu 35 identificações adicionais e uma economia de R$ 7.786,00 com a liberação do resultado positivo da cultura cinco dias à frente do ANC (VITEK 2). A redução do TAT pode contribuir para uma resolução clínica bem-sucedida.

Identification of Neural Stem Cell Biomarkers by Isobaric Tagging for Relative and Absolute Quantitation (iTRAQ) Mass Spectrometry.

This chapter describes a proteomic analysis of neural progenitor cells using isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry. A detailed procedure is described for the isolation, proliferation, and differentiation of these cells, including a comparative iTRAQ mass spectrometry analysis of the precursor and differentiated states. In total, there were changes in the levels of 55 proteins, many of which are not resolved easily by other proteomic methods. Therefore, this method should be useful for the identification of important regulatory molecules in the study of other precursor cells involved in neuronal or metabolic regulation in nutritional programming diseases.

Espectrometría de masas MALDI-TOF: evaluación de la etapa preanalítica para la identificación de hongos miceliales / MALDI-TOF mass spectrometry: Evaluation of the preanalytical phase for identification of molds

Se evaluaron 3 metodologías de extracción de proteínas para la identificación de hongos miceliales por MALDI-TOF MS en 44 aislados: la extracción con agua-ácido fórmico (E. agua), la extracción con zirconio-etanol-acetonitrilo-ácido fórmico (E. zirconio) y la recomendada por el proveedor del equipo (E. tubo). Se compararon 2 bases de datos: Bruker (BK) y BK + National Institutes of Health. Los resultados obtenidos utilizando dichas bases fueron los siguientes (en el orden citado): identificación correcta (IC) a nivel de género, 10 y 16 con E. agua; 27 y 32 con E. zirconio y 18 y 23 con E. tubo; IC a nivel de especie, 5 y 7 con E. agua; 17 y 20 con E. zirconio y 11 y 14 con E. tubo; identificaciones no confiables, 18 y 12 con E. agua y 9 y 4, tanto con E. zirconio como con E. tubo; ausencia de pico, 16 con E. agua, 8 con E. zirconio y 17 con E. tubo. La extracción con zirconio mostró el mejor rendimiento (p < 0,05).

In order to optimize the identification of molds with MALDI-TOF MS, three protein extraction-methodologies were evaluated against 44 isolates: water extraction (WE), zirconium extraction (ZE) and the provider's recommended method (PRM). Two data bases were compared, Bruker (BK) and Bruker + National Institutes of Health. Considering both databases, results were respectively as follows: correct identification (CI) at gender level, 10 and 16 by WE; 27 and 32 by ZE and 18 and 23 by PRM; CI at species level, 5 and 7 by WE; 17 and 20 by ZE and 11 and 14 by PRM; non-reliable identification, 18 and 12 by WE; 9 and 4 by ZE and by PRM. No peaks were observed in 16 by WE, 8 by ZE and 17 by PRM. ZE showed the best perfomance (p < 0.05).

The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification versus biochemical tests: a study with enterobacteria from a dairy cattle environment

Abstract Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n = 47) and fecal samples (n = 94) from cows, and samples from water (n = 23) and milk lines (n = 19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI-TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI-TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories.

[MALDI-TOF mass spectrometry: Evaluation of the preanalytical phase for identification of molds]. / Espectrometría de masas MALDI-TOF: evaluación de la etapa preanalítica para la identificación de hongos miceliales.

In order to optimize the identification of molds with MALDI-TOF MS, three protein extraction-methodologies were evaluated against 44 isolates: water extraction (WE), zirconium extraction (ZE) and the provider's recommended method (PRM). Two data bases were compared, Bruker (BK) and Bruker+National Institutes of Health. Considering both databases, results were respectively as follows: correct identification (CI) at gender level, 10 and 16 by WE; 27 and 32 by ZE and 18 and 23 by PRM; CI at species level, 5 and 7 by WE; 17 and 20 by ZE and 11 and 14 by PRM; non-reliable identification, 18 and 12 by WE; 9 and 4 by ZE and by PRM. No peaks were observed in 16 by WE, 8 by ZE and 17 by PRM. ZE showed the best perfomance (p<0.05).

The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification versus biochemical tests: a study with enterobacteria from a dairy cattle environment.

Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n=47) and fecal samples (n=94) from cows, and samples from water (n=23) and milk lines (n=19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI-TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI-TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories.

A metabolomic protocol for plant systematics by matrix-assisted laser-desorption/ionization time-of flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) has been widely used for the identification and classification of microorganisms based on their proteomic fingerprints. However, the use of MALDI-TOF MS in plant research has been very limited. In the present study, a first protocol is proposed for metabolic fingerprinting by MALDI-TOF MS using three different MALDI matrices with subsequent multivariate data analysis by in-house algorithms implemented in the R environment for the taxonomic classification of plants from different genera, families and orders. By merging the data acquired with different matrices, different ionization modes and using careful algorithms and parameter selection, we demonstrate that a close taxonomic classification can be achieved based on plant metabolic fingerprints, with 92% similarity to the taxonomic classifications found in literature. The present work therefore highlights the great potential of applying MALDI-TOF MS for the taxonomic classification of plants and, furthermore, provides a preliminary foundation for future research.

Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds.

The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, Trypsin-Sepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5 × 10(-8) and 3.0 × 10(-1) M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control.