Use of the DNA Checkerboard hybridization method for detection and quantitation of Candida species in oral microbiota

The DNA Checkerboard method enables the simultaneous identification of distinct microorganisms in a large number of samples and employs up to 45 whole genomic DNA probes to gram-negative and gram-positive bacterial species present in subgingival biofilms. Collectively, they account for 55%-60% of the bacteria in subgingival biofilms. In this study, we present the DNA Checkerboard hybridization as an alternative method for the detection and quantitation of Candida species in oral cavities. Our results reveal that DNA Checkerboard is sensitive enough and constitutes a powerful and appropriate method for detecting and quantifying Candida species found in the oral cavity.

Use of checkerboard DNA-DNA hybridization to evaluate the internal contamination of dental implants and comparison of bacterial leakage with cast or pre-machined abutments

To evaluate the checkerboard DNA-DNA hybridization method for detection and quantitation of bacteria from the internal parts of dental implants and to compare bacterial leakage from implants connected either to cast or to pre-machined abutments. Nine plastic abutments cast in a Ni-Cr alloy and nine pre-machined Co-Cr alloy abutments with plastic sleeves cast in Ni-Cr were connected to Branemark-compatible implants. A group of nine implants was used as control. The implants were inoculated with 3 mu l of a solution containing 10(8) cells/ml of Streptococcus sobrinus. Bacterial samples were immediately collected from the control implants while assemblies were completely immersed in 5 ml of sterile Tripty Soy Broth (TSB) medium. After 14 days of anaerobic incubation, occurrence of leakage at the implant-abutment interface was evaluated by assessing contamination of the TSB medium. Internal contamination of the implants was evaluated with the checkerboard DNA-DNA hybridization method. DNA-DNA hybridization was sensitive enough to detect and quantify the microorganism from the internal parts of the implants. No differences in leakage and in internal contamination were found between cast and pre-machined abutments. Bacterial scores in the control group were significantly higher than in the other groups (P < 0.05). Bacterial leakage through the implant-abutment interface does not significantly differ when cast or pre-machined abutments are used. The checkerboard DNA-DNA hybridization technique is suitable for the evaluation of the internal contamination of dental implants although further studies are necessary to validate the use of computational methods for the improvement of the test accuracy. To cite this article:do Nascimento C, Barbosa RES, Issa JPM, Watanabe E, Ito IY, Albuquerque Junior RF. Use of checkerboard DNA-DNA hybridization to evaluate the internal contamination of dental implants and comparison of bacterial leakage with cast or pre-machined abutments.Clin. Oral Impl. Res. 20, 2009; 571-577.doi: 10.1111/j.1600-0501.2008.01663.x.

Correlation of the in vitro antifungal drug susceptibility with the in vivo activity of fluconazole in a murine model of cerebral infection caused by Cryptococcus gattii

Forty Cryptococcus gattii strains were submitted to antifungal susceptibility testing with fluconazole, itraconazole, amphotericin B and terbinafine. The minimum inhibitory concentration (MIC) ranges were 0.5-64.0 for fluconazole, < 0.015-0.25 for itraconazole, 0.015-0.5 for amphotericin B and 0.062-2.0 for terbinafine. A bioassay for the quantitation of fluconazole in murine brain tissue was developed. Swiss mice received daily injections of the antifungal, and their brains were withdrawn at different times over the 14-day study period. The drug concentrations varied from 12.98 to 44.60 mu g/mL. This assay was used to evaluate the therapy with fluconazole in a model of infection caused by C. gattii. Swiss mice were infected intracranially and treated with fluconazole for 7, 10 or 14 days. The treatment reduced the fungal burden, but an increase in fungal growth was observed on day 14. The MIC for fluconazole against sequential isolates was 16 mu g/mL, except for the isolates obtained from animals treated for 14 days (MIC = 64 mu g/mL). The quantitation of cytokines revealed a predominance of IFN-gamma and IL-12 in the non-treated group and elevation of IL-4 and IL-10 in the treated group. Our data revealed the possibility of acquired resistance during the antifungal drug therapy.

Development and Validation of a Mice liar Electrokinetic Chromatography Method for Quantitative Determination of Butenolides in Piper malacophyllum (C. Presl) C. DC.

Introduction - A large number of natural and synthetic compounds having butenolides as a core unit have been described and many of them display a wide range of biological activities. Butenolides from P. malacophyllum have presented potential antifungal activities but no specific, fast, and precise method has been developed for their determination. Objective - To develop a methodology based on micellar electrokinetic chromatography to determine butenolides in Piper species. Methodology - The extracts were analysed in an uncoated fused-silica capillaries and for the micellar system 20 mmol/L SDS, 20% (v/v) acetonitrile (ACN) and 10 mmol/L STB aqueous buffer at pH 9.2 were used. The method was validated for precision, linearity, limit of detection (LOD) and limit of quantitation (LOQ) and the standard deviations were determined from the standard errors estimated by the regression line. Results - A micellar electrokinetic chromatography (MEKC) method for determination of butenolides in extracts gave full resolution for 1 and 2. The analytical curve in the range 10.0-50.0 mu g/mL (r(2) = 0.999) provided LOD and LOQ for 1 and 2 of 2.1/6.3 and 1.1/3.5 mu g/mL, respectively. The RSD for migration times were 0.12 and 1.0% for peak area ratios with 100.0 +/- 1.4% of recovery. Conclusions - A novel high-performance MEKC method developed for the analysis of butenolides 1 and 2 in leaf extracts of P. malacophyllum allowed their quantitative determined within an analysis time shorter than 5 min and the results indicated CE to be a feasible analytical technique for the quantitative determination of butenolides in Piper extracts. Copyright (C) 2010 John Wiley & Sons, Ltd.

Determination of amino acids by capillary electrophoresis-electrospray ionization-mass spectrometry: An evaluation of different protein hydrolysis procedures

In this work, a CE equipment, online hyphenated to an IT MS analyzer by a linear sheath liquid interface promoting ESI, was used to develop a method for quantitative determination of amino acids. Under appropriate conditions (BGE composition, 0.8% HCOOH, 20% CH(3)OH; sheath liquid composition, 0.8% HCOOH, 60% methanol; V(ESI), +4.50 W), analytical curves of all amino acids from 3 to 80 mg/L were recorded presenting acceptable linearity (r > 0.99). LODs in the range of 16-172 mu mol/L were obtained. BSA, a model protein, was submitted to different hydrolysis procedures (classical acid and basic, and catalyzed by the H(+) form of a cation exchanger resin) and its amino acid profiles determined. In general, the resin-mediated hydrolysis yields were overall similar or better than those obtained by classical acid or basic hydrolysis. The resulting experimental-to-theoretical BSA concentration ratios served as correction factors for the quantitation of amino acids in Brazil nut resin generated hydrolysates.

Fluoxetine and norfluoxetine analysis by direct injection of human plasma in a column switching liquid chromatographic system

A column switching LC method is presented for the analysis of fluoxetine (FLU) and norfluoxetine (NFLU) by direct injection of human plasma using a lab-made restricted access media (RAM) column. A RAM-BSA-octadecyl silica (C-18) column (40 min x 4.6 mm, 10 mu m) is evaluated in both backflush and foreflush elution modes and coupled with a C-18 lab-made (50 mm x 4.6 mm, 3 pm) analytical column in order to perform online sample preparation. Direct injection of 100 mu L, of plasma samples is possible with the developed approach. In addition, reduction of sample handling is obtained when compared with traditional liquid-liquid extraction (LLE) and SPE. The total analysis time is around 20 min. A LOQ of 15 ng/mL is achieved in a concentration range of 15-500 ng/mL, allowing the therapeutic drug monitoring of clinical samples. The precision values achieved are lower than 15% for all the evaluated points with adequate recovery and accuracy. Furthermore, no matrix interferences are found in the analysis and the proposed method shows to be an adequate alternative for analysis of FLU in plasma.

Matrix effects observed during pesticides residue analysis in fruits by GC

The influence of the sample matrix in the CC-electron-capture detection analysis of the pesticides dimethoate, diazinon, chlorothalonil.. parathion methyl and fenitrothion in fruits samples has been studied. Experiments have been carried out where the pesticide responses in standard solutions prepared in selected solvent were compared with their response when present in apple, mango, papaya, banana, pineapple and melon extracts. The presence of matrix effects (MEs) and their extent were shown to be simultaneously influenced by several factors (matrix concentration, matrix type, pesticide concentration, analytical range). Pronounced MEs were observed particularly for dimethoate and diazinon in all matrices tested; in lower concentrations, all pesticides presented significant ME. The other pesticides presented variable ME. Higher ME enhancement was detected at lower pesticide concentration levels of and/or at higher matrix concentration solutions. The ME detected for fenitrothion, in the analytical range evaluated, were dependent on matrix type. For each pesticide, solvent and matrix-matched calibrations were compared for all fruit samples, and it could be concluded that quantitation based on standard solutions prepared in blank matrix extract (matrix-matched calibration) should be used to compensate the MEs and to obtain more accurate results for the pesticides studied.