Relationship between gingival clinical parameters and the reactivity of the BANA test in subgingival samples from children.

Gingivitis is the first manifestation of periodontal disease, and is characterized by painless and slow evolution. Early diagnosis and intervention must be done to avoid the possibility of precocious periodontitis during the childhood or teenage years. The enzymatic BANA test (N-benzoyl-DL-arginine-naphthylamide) was used to evaluate subgingival samples from 54 children between 6 and 9 years of age. Plaque index (PI) and gingival index (GI) were assessed according to the criteria recommended by Löe (1967). Subgingival plaque was collected from the region that featured the greatest periodontal alteration, represented by a higher gingival index. Resulting data were grouped individually according to visible and non-visible plaque and bleeding and non-bleeding gingiva. Results showed that there was no statistically significant correlation between the presence of visible plaque and the positivity of the BANA test, nor was there a statistically significant correlation between the presence of bleeding and the positivity of the BANA test in subgingival samples obtained from children. This study concluded that the BANA test is not an ideal diagnostic test to be applied to children.

Toxoplasma gondii: Detection by mouse bioassay, histopathology, and polymerase chain reaction in tissues from experimentally infected pigs

In the present study, we evaluated three techniques, mouse bioassay, histopathology, and polymerase chain reaction (PCR) to detect Toxoplasma gondii infection in tissues from experimentally infected pigs. Twelve mixed breed pigs, seronegative for T. gondii using an indirect immunofluorescent antibody test (IFAT), were used. Ten pigs were infected with 4 × 104 VEG strain oocysts, and two were maintained as uninfected controls. Animals were killed 60 days pos infection. Muscle (heart, tongue, diaphragm, and masseter) and brain samples were collected to investigate the presence of T. gondii tissue cysts by the different assay methods. For the bioassay, samples of brain (50 g) and pool of muscle samples (12.5 g of tongue, masseter, diaphragm, and heart) were used. PCR was performed using Tox4 and Tox5 primers which amplified a 529 bp fragment. The DNA extraction and PCR were performed three times, and all tissue samples were tested individually (brain, tongue, masseter, diaphragm, and heart). For histopathology, fragments of tissues were fixed in 10% of buffered formal saline and stained with HE. Histopathological results were all negative. PCR showed 25/150 (16.6%) positive samples, being 17/120 (14.1%) and 8/30 (26.6%) from muscle, and brain tissues, respectively. Tissue cysts of T. gondii were identified by mouse bioassay in 54/98 (55.1%) samples, being 31/48 (64.6%) from muscle samples, and 23/50 (46.0%) from brain samples. Toxoplasma gondii isolation in muscle samples by mouse bioassay was higher than in PCR (P < 0.01). Results indicate that DNA from pig tissues interfered with 529-bp-PCR sensitivity, and mouse bioassay was better than PCR in detecting T. gondii in tissues from pigs. © 2006 Elsevier Inc. All rights reserved.

Rapid molecular diagnosis of tuberculosis and other mycobacteriosis in smear-negative clinical specimens

Delay in diagnosis of pulmonary and other forms of tuberculosis (TB) can be fatal, particularly in HIV-infected patients. Hence, techniques based on nucleic acid amplification, which are both rapid and of high specificity and sensitivity, are now widely used and recommended for laboratories that diagnose TB. In the present study, diagnostic methods based on mycobacterial DNA amplification were evaluated in comparative trials alongside tradicional bacterial methods, using negative smear samples from patients with clinically-suspected TB (sputum samples from 25 patients with suspected pulmonary TB, urine samples from two patients with suspected renal TB and cerebrospinal fluid samples from one patient with suspected meningeal TB). A specificity of 100% was achieved with DNA amplification methods and tradicional culture/identification methods, in relation to clinical findings and treatment results. For the smear-negative sputa, conventional PCR for M. tuberculosis was positive in 62% of suspected lung TB case, showing the same sensitivity as bacterial identification. Both techniques failed in the detection of extra-pulmonary samples. Nested PCR showed, after species-specific amplification, a sensitivity of 100% for M. avium and 85% for M. tuberculosis. For extra-pulmonary smear-negative samples, only Nested PCR detected M. tuberculosis and all cases were confirmed clinically. Nested PCR, in which two-step amplification reactions are performed, can identify the two most important mycobacteria in human pathology quickly and directly from clinical spicimens.

Microdon tigrinus (Diptera, Syrphidae): Populational fluctuations and specificity to the nest of Acromyrmex coronatus (Hymenoptera: Formicidae)

This study was performed in the southern and southeastern regions of Brazil, visiting numerous municipalities, over a period of 25 years, excavating nests of 12 species of leaf-cutting ants of the Acromyrmex genus. Larvae and pupae of Microdon tigrinus were found only in nests of the leaf-cutting ant Acromyrmex coronatus, indicating high specificity. Observation showed that larvae and pupae were well accepted in the nests and the adults, immediately after puparia eclosion and prior to wing distension, were not attacked by the workers, suggesting that they produce semiochemicals for a short time period until they arrive outside the Acromyrmex coronatus nest. It was postulated that these larvae feed on the organic detritus of the nest, as shown for Microdon larvae of other species.

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G1 (infected group, n = 10) and G2 (uninfected group, n = 8). Infection was performed with 4 × 10 4 VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T. gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T. gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T. gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) × IFA (ah) (r = 0.62, P = 0.05), MAT(s) × MAT (ah) (r = 0.97, P < 0.0001); however, there was no significant difference between r-ELISA(s) × r-ELISA (ah) (r = 0.14, P = 0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis. © 2007 Elsevier Ltd. All rights reserved.

Polyethylene gel in the subcutaneous tissue of rats: Histopathologic and systemic evaluation

Purpose: To evaluate the histological and systemic response to subcutaneous injection of polyethylene gel in rats. Methods: Twenty-one white male rats were divided into 3 groups (G): G1 and G2 received subcutaneous polyethylene gel injection in the dorsal midline and were sacrificed at 30 and 60 postoperative days, respectively. G3 was not exposed to the polyethylene gel and was sacrificed after 60 days. Blood levels of lactate dehydrogenase (LDH), creatine kinase (CK), and alkaline phosphatase (ALP) were evaluated. The heart, kidney, liver, adrenal gland, injection site, and adjacent tissues were histologically examined. The results were submitted to statistical analysis. Results: There was no clinical evidence of extrusion, reduction of the injected volume, or abnormalities in the adjacent tissues. Blood levels of CK and LDH were normal and similar in all groups. ALP levels were significantly lower in G2 than in G1 and G3. The systemic organs were normal on histological examination in the 3 groups evaluated. Microscopically, the polyethylene gel was surrounded by a thin pseudocapsule formation and minimal inflammatory cell response, which decreased from G1 to G2. Conclusion: The subcutaneous injection of polyethylene gel in rats elicited minimal local inflammatory response and no systemic side effects. Copyright © 2008 Informa Healthcare USA, Inc.

Influence of lower cutoff values for 100-g oral glucose tolerance test and glycemic profile for identification of pregnant women at excessive fetal growth risk

Objective: To evaluate data from patients with normal oral glucose tolerance test (OGTT) results and a normal or impaired glycemic profile (GP) to determine whether lower cutoff values for the OGTT and GP (alone or combined) could identify pregnant women at risk for excessive fetal growth. Methods: We classified 701 pregnant women with positive screening for gestational diabetes mellitus (GDM) into 2 categories - (1) normal 100-g OGTT and normal GP and (2) normal 100-g OGTT and impaired GP - to evaluate the influence of lower cutoff points in a 100-g OGTT and GP (alone or in combination) for identification of pregnant women at excessive fetal growth risk. The OGTT is considered impaired if 2 or more values are above the normal range, and the GP is impaired if the fasting glucose level or at least 1 postprandial glucose value is above the normal range. To establish the criteria for the OGTT (for fasting and 1, 2, and 3 hours after an oral glucose load, respectively), we considered the mean (75 mg/dL, 120 mg/dL, 113 mg/dL, and 97 mg/dL), mean plus 1 SD (85 mg/dL, 151 mg/dL, 133 mg/dL, and 118 mg/dL), and mean plus 2 SD (95 mg/dL, 182 mg/dL, 153 mg/dL, and 139 mg/dL); and for the GP, we considered the mean and mean plus 1 SD (78 mg/dL and 92 mg/dL for fasting glucose levels and 90 mg/dL and 130 mg/dL for 1- or 2-hour postprandial glucose levels, respectively). Results: Subsequently, the women were reclassified according to the new cutoff points for both tests (OGTT and GP). Consideration of values, in isolation or combination, yielded 6 new diagnostic criteria. Excessive fetal growth was the response variable for analysis of the new cutoff points. Odds ratios and their respective confidence intervals were estimated, as were the sensitivity and specificity related to diagnosis of excessive fetal growth for each criterion. The new cutoff points for the tests, when used independently rather than collectively, did not help to predict excessive fetal growth in the presence of mild hyperglycemia. Conclusion: Decreasing the cutoff point for the 100-g OGTT (for fasting and 1, 2, and 3 hours) to the mean (75 mg/dL, 120 mg/dL, 113 mg/dL, and 97 mg/dL) in association with the GP (mean or mean plus 1 SD-78 mg/dL and 92 mg/dL for the fasting state and 90 mg/dL and 130 mg/dL for 1- or 2-hour postprandial values-increased the sensitivity and specificity, and both criteria had statistically significant predictive power for detection of excessive fetal growth. © 2008 AACE.

A Bayesian approach to estimate the accuracy of in-house ELISA assay to measure rabies antibodies from compulsory vaccinated dogs and cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Development and application of a highly selective biomimetic sensor for detection of captopril, an important ally in hypertension control

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

Detection of SPM and IMP metallo-β-lactamases in clinical specimens of Pseudomonas aeruginosa from a Brazilian public tertiary hospital

Phenotypic and genotypic SPM and IMP metallo-β-lactamases (MBL) detection and also the determination of minimal inhibitory concentrations (MIC) to imipenem, meropenem and ceftazidime were evaluated in 47 multidrug-resistant Pseudomonas aeruginosa isolates from clinical specimens. Polymerase chain reaction detected 14 positive samples to either blaSPM or blaIMP genes, while the best phenotypic assay (ceftazidime substrate and mercaptopropionic acid inhibitor) detected 13 of these samples. Imipenem, meropenem and ceftazidime MICs were higher for MBL positive compared to MBL negative isolates. We describe here the SPM and IMP MBL findings in clinical specimens of P. aeruginosa from the University Hospital of Botucatu Medical School, São Paulo, Brazil, that reinforce local studies showing the high spreading of blaSPM and blaIMP genes among Brazilian clinical isolates. © 2011 Elsevier Editora Ltda.

Use of reverse transcriptase polymerase chain reaction (RT-PCR) in molecular screening of Newcastle disease virus in poultry and free-living bird populations

The aim of this study was to evaluate a simple molecular method of reverse transcriptase polymerase chain reaction (RT-PCR) to differentiate Newcastle disease virus strains according to their pathogenicity, in order to use it in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Specific primers were developed to differentiate LaSota-LS-(vaccine strain) and Sao Joao do Meriti-SJM-strain (highly pathogenic strain). Chickens and pigeons were experimentally vaccinated/infected for an in vivo study to determine virus shedding in feces. Validation of sensitivity and specificity of the primers (SJM and LS) by experimental models used in the present study and results obtained in the molecular analysis of the primers by BLAST made it possible to generalize results. The development of primers that differentiate the level of pathogenicity of NDV stains is very important, mainly in countries where real-time RT-PCR is still not used as a routine test. These primers were able to determine the presence of the agent and to differentiate it according to its pathogenicity. © 2012 Springer Science+Business Media B.V.

Traditional and novel methods for occlusal caries detection: Performance on primary teeth

This study aimed to assess the performance of International Caries Detection and Assessment System (ICDAS), radiographic examination, and fluorescence-based methods for detecting occlusal caries in primary teeth. One occlusal site on each of 79 primary molars was assessed twice by two examiners using ICDAS, bitewing radiography (BW), DIAGNOdent 2095 (LF), DIAGNOdent 2190 (LFpen), and VistaProof fluorescence camera (FC). The teeth were histologically prepared and assessed for caries extent. Optimal cutoff limits were calculated for LF, LFpen, and FC. At the D 1 threshold (enamel and dentin lesions), ICDAS and FC presented higher sensitivity values (0.75 and 0.73, respectively), while BW showed higher specificity (1.00). At the D 2 threshold (inner enamel and dentin lesions), ICDAS presented higher sensitivity (0.83) and statistically significantly lower specificity (0.70). At the D 3 threshold (dentin lesions), LFpen and FC showed higher sensitivity (1.00 and 0.91, respectively), while higher specificity was presented by FC (0.95), ICDAS (0.94), BW (0.94), and LF (0.92). The area under the receiver operating characteristic (ROC) curve (Az) varied from 0.780 (BW) to 0.941 (LF). Spearman correlation coefficients with histology were 0.72 (ICDAS), 0.64 (BW), 0.71 (LF), 0.65 (LFpen), and 0.74 (FC). Inter- and intraexaminer intraclass correlation values varied from 0.772 to 0.963 and unweighted kappa values ranged from 0.462 to 0.750. In conclusion, ICDAS and FC exhibited better accuracy in detecting enamel and dentin caries lesions, whereas ICDAS, LF, LFpen, and FC were more appropriate for detecting dentin lesions on occlusal surfaces in primary teeth, with no statistically significant difference among them. All methods presented good to excellent reproducibility. © 2012 Springer-Verlag London Ltd.

Purification, characterization, and specificity determination of a new serine protease secreted by penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl2, KCl, and BaCl, and partially inhibited by CuCl2, CoCl2 and totally inhibited by AlCl3 and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k cat/K m of 10,666 mM-1 s-1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k cat/K m of 7,500 mM -1 s-1. Basic and acidic side chain-containing amino acids performed best at subsite S1. Subsites S2, S3, S′ 2, and S′ 1, S ′ 3 showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k cat/K m were observed for the subsites S2, S3, and S′ 2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level. © 2012 Springer Science+Business Media New York.

Detection of cavitated approximal surfaces using cone beam CT and intraoral receptors

Objectives: The aim of this study was to compare cone beam CT (CBCT) in a small field of view (FOV) with a solid-state sensor and a photostimulable phosphor plate system for detection of cavitated approximal surfaces. Methods: 257 non-filled approximal surfaces from human permanent premolars and molars were recorded by two intraoral digital receptors, a storage phosphor plate (Digora Optime, Soredex) and a solid-state CMOS sensor (Digora Toto, Soredex), and scanned in a cone beam CT unit (3D Accuitomo FPD80, Morita) with a FOV of 4 cm and a voxel size of 0.08 mm. Image sections were carried out in the axial and mesiodistal tooth planes. Six observers recorded surface cavitation in all images. Validation of the true absence or presence of surface cavitation was performed by inspecting the surfaces under strong light with the naked eye. Differences in sensitivity, specificity and agreement were estimated by analysing the binary data in a generalized linear model using an identity link function. Results: A significantly higher sensitivity was obtained by all observers with CBCT (p,0.001), which was not compromised by a lower specificity. Therefore, a significantly higher overall agreement was obtained with CBCT (p,0.001). There were no significant differences between the Digora Optime phosphor plate system and the Digora Toto CMOS sensor for any parameter. Conclusions: CBCT was much more accurate in the detection of surface cavitation in approximal surfaces than intraoral receptors. The differences are interpreted as clinically significant. A CBCT examination performed for other reasons should also be assessed for approximal surface cavities in teeth without restorations. © 2013 The British Institute of Radiology.

Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)