Research on Candida dubliniensis in a Brazilian yeast collection obtained from cardiac transplant, tuberculosis, and HIV-positive patients, and evaluation of phenotypic tests using agar screening methods

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A label-free immunosensor based on recordable compact disk chip for early diagnostic of the dengue virus infection

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Trypanosoma cruzi: identification and characterization of a novel ribosomal protein L27 (TcrL27) that cross-reacts with an affinity-purified anti-Sm antibody

Small nuclear ribonucleoproteins (snRNPs)are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.

Detection and quantification of antibodies to Newcastle disease virus in ostrich and rhea sera using a liquid phase blocking enzyme-linked immunosorbent assay

A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (Struthio camelus) and rheas (Rhea americana) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutination-inhibition (HI) test was regarded as the reference method, the cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (P < 0.0001) on the HI titers with a high correlation coefficient (r = 0.875). The two tests showed good agreement ( = 0.82; P < 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable.

Recommended Guidelines for Submission, Trimming, Margin Evaluation, and Reporting of Tumor Biopsy Specimens in Veterinary Surgical Pathology

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.

Chronic Suppurative Otitis Media in Cleft Palate: Microorganism Etiology and Susceptibilities

Objective: To investigate the microbial etiology of suppurative chronic otitis media (SCOM) in patients with complete cleft lip and palate and isolated cleft palate and to determine the sensitivity of isolated microorganisms to antibiotics by drug diffusion from impregnated discs in agar and the minimum inhibitory concentration of each drug to these microorganisms by drug dilution in agar.Design/Patients: Effusion samples of SCOM obtained from 40 patients with cleft lip and palate registered at the Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo, at Bauru, Brazil, were bacteriologically analyzed by cultures. The isolated bacteria were submitted to an in vitro susceptibility test to clinically used drugs.Results: Positive cultures were obtained in 100% of studied cases. Among the 57 strains observed, the most frequent were Pseudomonas aeruginosa (35%), Staphylococcus aureus (15.5%), Enterococcus faecalis (14%), and Proteus mirabilis (12%). The frequency of Gram-negative bacilli (enterobacteriaceae and nonfermentative bacilli) was 67%. Pseudomonas aeruginosa presented the highest sensitivity to ciprofloxacin, and enterobacteriaceae exhibited the highest sensitivity to gentamicin. The strains of S. aureus and E. faecalis presented the highest sensitivity to imipenem and sulfamethoxazole/trimethoprim, respectively.Conclusion: Patients with cleft lip and palate presenting with SCOM exhibited 100% positive cultures, with the highest frequency of Pseudomonas and enterobacteriaceae. With regard to the action of antibiotics, imipenem was effective against the four species of isolated microorganisms, followed by ciprofloxacin, which was effective against 75% of isolated species.

Rat nephrotoxic nephritis. The relation between the type and intensity of induced histological lesions and the content of antiglomerular basement membrane antibodies of the inoculated serum

Six groups of 6 rats received equal doses (0.8 ml/100 g of body weight) of different rabbit anti rat kidney sera. The titer of anti GBM antibodies in the sera was evaluated by indirect immunofluorescent test in isolated GBM (IIT GBM). Rats of groups 1, 2, 3, 5, 6 received anti rat GBM sera with titers of 1/320, 1/240, 1/160, 1/60, 1/30 respectively. Group 4 received anti rat kidney serum with a titer of 1/80. The rats of group 1 died from 1 to 5 minutes after inoculation and their kidney were congested, with hialine trombi occluding arterioles and glomerular capillaries. The rats of group 2 and one of group 3 died from 2 to 15 days after inoculation and diffuse cortical necrosis was found. The remaining rats were sacrificed 2 months after inoculation. The kidneys were normal in control group; chronic membranoproliferative glomerulonephritis was observed in group 3 and 4, membranoproliferative glomerulonephritis in group 5 and minimal changes in group 6. By immunofluorescence rabbit gammaglobulin was seen in GBM of group 3, 4, 5 and 6. The IIT GBM performed in the eluates of the kidneys revealed the presence of heterologous antibody in groups 1, 2, 3, 4, 5 and 6 and autologous antibody in groups 3, 4 and 5. One concludes that the IIT GBM identifies and quantifies antibodies which have the property of damaging the kidney.

Epidemiology and genetics of endemic goiter. II. Genetic aspects

Complex genetic models and segregation analysis were applied to family data obtained in a hyperendemic goiter area in Brazil. The single locus and Falconer's models did not fit the data. Edward's model showed convergency, but statistical concordance has not been obtained. Although the genetic load model explains statistically the family data, it would be hard to imagine that endemic goiter could be explained by a model where synergism among genetic and environmental factors is not assumed.

Prevalence and severity of caries in 3-12-year-old children from three districts with different fluoridation histories in Araraquara, SP, Brazil

Objective: To determine the prevalence and severity of caries in 3-12-year-old children in three districts of the city of Araraquara, Design: An epidemiological survey was carried out by trained and calibrated examiners in 1995 and 1996, using the WHO diagnostic criteria. Setting: Two districts, Araraquara and Vila Xavier had been fluoridated since 1963 and one, Gavião Peixoto, since 1994. Subjects: The study included 1,191 children from Araraquara, 653 from Vila Xavier and 652 from Gavião Peixoto. Outcome measures: Prevalence of caries, dmft, dmfs, DMFT and DMFS indices. Results: Results showed moderate caries experience in all three districts. Differences between districts in relation to fluoridation history were particularly obvious in primary teeth. In 3-4-year-old children, one third of those in Araraquara and Vila Xavier had some caries experience compared to 58% in Gavião Peixoto. In permanent teeth, 20% or less of the mean DMFT was made up of untreated decay in Araraquara and Vila Xavier whereas in Gaviao Peixoto it made up between 50 and 57% of values in 7-12-year-old children. Conclusions: The prevalence and severity of caries was lower in dentitions of children from the districts fluoridated since 1963. Improvements are likely in the future in Gaviao Peixoto as the benefit of fluoridation continues but additional means of promoting oral health are needed in all three districts.

Laparoscopic evaluation and treatment of the impalpable testis

Objectives: To evaluate the laparoscopic technique as a diagnostic and therapeutic tool in the management of patients with impalpable testis. Material and Methods: Fifty-nine patients with mean age of 6.3 years underwent laparoscopy to evaluate 85 impalpable testes that were classified as absent, canalicular and intra-abdominal. In the case of testicular absence, the procedure was terminated. In the case of canalicular testis, open inguinal exploration was performed. In intra-abdominal testis, either laparoscopic orchiopexy or orchiectomy was performed. According to the length of the vascular pedicle, orchipexy was performed either with or without vascular ligature. Post-operatively, the treated testes were evaluated according to size and location in the scrotum. Results: Seventeen (20%) of the 85 impalpable testes were diagnosed as absent, 21 (24.7%) as canalicular and 47 (55.3%) as intra-abdominal. Of the canalicular testes, 20 were explored by inguinotomy and one by laparoscopy. All the intra-abdominal testes were treated initially by laparoscopy, four being removed due to atrophy, 31 submitted to vascular ligature and 12 to primary orchipexy. Of those submitted to vascular ligature, 22 underwent a second stage orchipexy, of which 18 laparoscopically and 4 by inguinotomy. Of the 18 testes brought to the scrotum by staged laparoscopic orchipexy, 15 (83.3%) presented normal characteristics in the late follow-up, while of the 12 submitted to primary laparoscopic orchipexy, 8 (66.6%) were normal. There were no perioperative or late complications. Conclusions: Laparoscopy is a minimally invasive procedure with low morbidity that enables precise diagnosis of the impalpable testes. When intra-abdominal testes are found, either immediate laparoscopic orchiectomy, or primary and staged orchipexy are possible, with results equivalent to open procedures, with the advantage of smaller surgical incisions and shorter postoperative recovery.

Somatic meiosis and development of the juvenile gametophyte in members of the Batrachospermales sensu lato (Rhodophyta)

Seven populations (six in culture and one sampled directly from nature) of the freshwater red algal families Batrachospermaceae, Lemaneaceae and Thoreaceae were examined, involving three species of Batrachospermum, two of Paralemanea and one of Thorea. All 'Chantransia' stages ultimately produced juvenile gametophytes. The production of juvenile gametophytes in the three populations of Batrachospermum was generally most abundant at 15°C and low irradiances (47-68 μmol photons m-2 s-1). The most abundant gametophyte development in the Paralemanea species was observed at 10°C and low or high irradiances (47-142 μmol photons m-2 s-1). Gametophyte production in Thoreaceae occurred at higher temperatures (20°C) and also at low irradiances. In species of the Batrachospermaceae and Lemaneaceae, the 'elimination cells' can be situated on the basal or suprabasal cell of the juvenile gametophyte, but the position is usually fixed in individual species. The presence and position of the elimination cells remain to be established in Thoreaceae. Our results corroborate a previous study suggesting that the position of elimination cells is such a constant feature that it is of potential diagnostic value at the generic or infrageneric (sectional or specific) level. The characteristics observed in the development of the juvenile gametophytes in species of Batrachospermaceae and Lemaneaceae essentially agreed with general descriptions in the previous studies. The characteristics of the Thoreaceae, with a distinctive developmental pattern of the juvenile gametophyte and the occurrence of two morphological types in the 'Chantransia' stage, support the proposal to elevate it to the ordinal level. Two remarkable observations in Batrachospermum species were the production of numerous juvenile gametophytes from filaments of the same plant of the 'Chantransia' stage and the formation of a system of rhizoidal filaments or cell agglomeration of the juvenile gametophytes, which produced new gametophytes. These two characteristics potentially increase the formation of additional gametophytes under favourable conditions.

Redescription and reevaluation of the generic status of Leptodactylus dantasi (Amphibia, Anura, Leptodactylidae) and description of its unusual advertisement call

Leptodactylus dantasi Bokermann is redescribed from adult specimens collected in Parque Nacional da Serra do Divisor, State of Acre, Brazil, near the border between the Brazilian state of Acre and Peru. We propose the inclusion of this species in the genus Hydrolaetare. A new diagnostic character observed for the genus is the presence of fringes of fingers, and fringes and webbing of toes, finely serrate; the serrate edge of the fringe and webbing can be keratinized in males, females, and subadults. Hydrolaetare dantasi (Bokermann) is characterized by robust body and limbs, a broad and depressed head, slightly shorter than wide, and long, pointed and basally webbed toes. Hydrolaetare dantasi differs from the only other species in this genus, Hydrolaetare schmidti (Cochran and Goin), mainly by having toes webbed basally (fully webbed in H. schmidti). The advertisement call of H. dantasi is composed of two components; an initial note produced by the impact of the vocal sac against the ground and a second note corresponding to a long whistle of ascending frequency. The initial note is a percussive sound and represents an unusual form of sound emission in anurans; the second note is a vocalization.

Development and validation of HPLC method for quantitative analysis of triamcinolone in biodegradable microparticles

A simple, rapid, selective and specific high performance liquid chromatographic (HPLC) method for quantitative analysis of the triamcinolone in polylactide-co-glycolide acid (PLGA) microparticles was developed. The chromatographic parameters were reversed-phase C18 column, 250mm x 4.6mm, with particle size 5 μm. The column oven was thermostated at 35°C ± 2°C. The mobile phase was methanol/water 45:55 (v/v) and elution was isocratic at a flow-rate of 1 mL.mL-1. The determinations were performed using a UV-Vis detector at 239 nm. The injected sample volume was 10 μL. The standard curve was linear (r2 > 0.999) in the concentration range 100-2500 ng.mL-1. The method showed adequate precision, with a relative standard deviation (RSD) was smaller than 3%. The accuracy was analyzed by adding a standard drug and good recovery values were obtained for all drug concentrations used. The method showed specificity and selectivity with linearity in the working range and good precision and accuracy, making it very suitable for quantitation of triamcinolone in PLGA microparticles.

Doppler ultrasonography principles and methods of evaluation of the reproductive tract in mares

Background: Doppler ultrasonography is a non-invasive real time pulse-wave technique recently used for the transrectal study of the reproductive system hemodynamics in large animals. This technic is based in the Doppler Effect Principle that proposes the change in frequency of a wave for an observer (red blood cells) moving relative to the source of the respective wave (ultrasonic transducer). This method had showed to be effective and useful for the evaluation of the in vivo equine reproductive tract increasing the diagnostic, monitoring, and predictive capabilities of theriogenology in mares. However, an accurate and truthful ultrasonic exam requires the previous knowledge of the Doppler ultrasonography principles. Review: In recent years, the capabilities of ultrasound flow imaging have increased enormously. The current Doppler ultrasound machines offer three methods of evaluation that may be used simultaneously (triplex mode). In B-mode ultrasound, a linear array of transducers simultaneously scans a plane through the tissue that can be viewed as a two-dimensional gray-scale image on screen. This mode is primarily used to identify anatomically a structure for its posterior evaluation using colored ultrasound modes (Color or Spectral modes). Colored ultrasound images of flow, whether Color or Spectral modes, are essentially obtained from measurements of moving red cells. In Color mode, velocity information is presented as a color coded overlay on top of a B-mode image, while Pulsed Wave Doppler provides a measure of the changing velocity throughout the cardiac cycle and the distribution of velocities in the sample volume represented by a spectral graphic. Color images conception varies according to the Doppler Frequency that is the difference between the frequency of received echoes by moving blood red cells and wave frequency transmitted by the transducer. To produce an adequate spectral graphic it is important determine the position and size of the simple gate. Furthermore, blood flow velocity measurement is influence by the intersection angle between ultrasonic pulses and the direction of moving blood-red cells (Doppler angle). Objectively colored ultrasound exam may be done on large arteries of the reproductive tract, as uterine and ovary arteries, or directly on the target tissue (follicle, for example). Mesovarium and mesometrium attachment arteries also can be used for spectral evaluation of the equine reproductive system. Subjectively analysis of the ovarian and uterine vascular perfusion must be done directly on the corpus luteum, follicular wall and uterus (endometrium and myometrium associated), respectively. Power-flow imaging has greater sensitivity to weak blood flow and independent of the Doppler angle, improving the evaluation of vessels with small diameters and slow blood flow. Conclusion: Doppler ultrasonography principles, methods of evaluation and reproductive system anatomy have been described. This knowledge is essential for the competent equipment acquisition and precise collection and analysis of colored ultrasound images. Otherwise, the reporting of inconsistent and not reproducible findings may result in the discredit of Doppler technology ahead of the scientific veterinary community.