The role of P16ink4a and P53 immunostaining in predicting recurrence of HG-CIN after conization treatment

Objective: Io evaluate the expression of p16INK4a and p53 biomarkers in conization specimens from patients with high grade cervical intraepithelial neoplasia (HG-CIN), correlating them with the ability to predict the recurrence. Methods : we conducted a retrospective study of patients with HG-CIN in cervical biopsy treated with conization between January 1999 and January 2006 who had a minimum follow-up of 18 months. The expression of the p16 and p53 was assessed by tissue microarrays and correlated with disease recurrence. For analysis, we used the test of proportions (chi-square), considering value p<0.05, 95% CI and calculations of sensitivity, specificity and accuracy of these immunomarkers in predicting recurrence. Results : the series comprised 83 patients aged between 16 and 86 years (35±11.7), divided into two groups: 30 with HG-CIN recurrence (study group) and 53 without recurrence (control group). Mean age, parity, smoking and conization technique were similar in both groups. The p53 expression was present in 43% of the study group and 57% of the control group, and the p16 was present in 43% of the study group and in 57% of the control group (p>0.05). p53 had a positive predictive value (PPV) of 42% and negative predictive value (NPV) of 73%, sensitivity 70%, specificity of 47% and accuracy of 59%. The p16, PPV 42%, NPV 72%, sensitivity 66%, specificity of 49% and accuracy of 56%. Conclusion : immunohistochemistry expression of p53 and p16 showed low sensitivity and low specificity as predictors of HG-CIN recurrence after conization treatment.

Quantification of Trypanosoma cruzi in the heart, lymph nodes and liver of experimentally inffected mice, using limiting dilution analysis

Limiting dilution analysis was used to quantify Trypanosoma cruzi in the lymph nodes, liver and heart of Swiss and C57 B1/10 mice. The results showed that, in Swiss and B1/10 mice infected with T. cruzi Y strain, the number of parasites/mg of tissue increased during the course of the infection in both types of mice, although a grater number of parasites were observed in heart tissue from Swiss mice than from B1/10. With regard to liver tissue, it was observed that the parasite load in the initial phase of infection was higher than in heart. In experiments using T. cruzi Colombian strain, the parasite load in the heart of Swiss and B1/10 mice increased relatively slowly, although high levels of parasitization were nonetheless observable by the end of the infection. As for the liver and lymph nodes, the concentration of parasites was lower over the entire course of infection than in heart. Both strains thus maintained their characteristic tissue tropisms. The limiting dilution assay (LDA) proved to be an appropriate method for more precise quantification of T. cruzi, comparing favorably with other direct microscopic methods that only give approximate scores.

Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV) RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+), HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+), acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-). All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection.

MERCURY QUANTIFICATION IN SOILS USING THERMAL DESORPTION AND ATOMIC ABSORPTION SPECTROMETRY: PROPOSAL FOR AN ALTERNATIVE METHOD OF ANALYSIS

Despite the considerable environmental importance of mercury (Hg), given its high toxicity and ability to contaminate large areas via atmospheric deposition, little is known about its activity in soils, especially tropical soils, in comparison with other heavy metals. This lack of information about Hg arises because analytical methods for determination of Hg are more laborious and expensive compared to methods for other heavy metals. The situation is even more precarious regarding speciation of Hg in soils since sequential extraction methods are also inefficient for this metal. The aim of this paper is to present a technique of thermal desorption associated with atomic absorption spectrometry, TDAAS, as an efficient tool for quantitative determination of Hg in soils. The method consists of the release of Hg by heating, followed by its quantification by atomic absorption spectrometry. It was developed by constructing calibration curves in different soil samples based on increasing volumes of standard Hg2+ solutions. Performance, accuracy, precision, and quantification and detection limit parameters were evaluated. No matrix interference was detected. Certified reference samples and comparison with a Direct Mercury Analyzer, DMA (another highly recognized technique), were used in validation of the method, which proved to be accurate and precise.

Comparison of USEPA 3050B and ISO 14869-1: 2001 digestion methods for sediment analysis by using FAAS and ICP-OES quantification techniques

A study of the partial USEPA 3050B and total ISO 14869-1:2001 digestion methods of sediments was performed. USEPA 3050B was recommended as the simpler method with less operational risk. However, the extraction ability of the method should be taken in account for the best environmental interpretation of the results. FAAS was used to quantify metal concentrations in sediment solutions. The alternative use of ICP-OES quantification should be conditioned by a previous detailed investigation and eventual correction of the matrix effect. For the first time, the EID method was employed for the detection and correction of the matrix effect in sediment ICP-OES analysis. Finally, some considerations were made about the level of metal contamination in the area under study.

Analysis of passion fruit rinds (Passiflora edulis): isoorientin quantification by HPTLC and evaluation of antioxidant (radical scavenging) capacity

The content of isoorientin in passion fruit rinds (Passiflora edulis fo. flavicarpa O. Degener) was determined by HPTLC (high performance thin layer chromatography) with densitometric analysis. The results revealed a higher amount of isoorientin in healthy rinds of P. edulis (92.275 ± 0.610 mg L-1) than in rinds with typical symptoms of PWV (Passion fruit Woodiness Virus) infection (28.931 ± 0.346 mg L-1). The HPTLC data, allied to assays of radical scavenging activity, suggest the potential of P. edulis rinds as a natural source of flavonoids or as a possible functional food.

A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping

Introduction Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5′ untranslated region (5′UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping.

Cerebral perfusion and automated individual analysis using SPECT among an obsessive-compulsive population

OBJECTIVE: To make individual assessments using automated quantification methodology in order to screen for perfusion abnormalities in cerebral SPECT examinations among a sample of subjects with OCD. METHODS: Statistical parametric mapping (SPM) was used to compare 26 brain SPECT images from patients with OCD individually with an image bank of 32 normal subjects, using the statistical threshold of p < 0.05 (corrected for multiple comparisons at the level of individual voxels or clusters). The maps were analyzed, and regions presenting voxels that remained above this threshold were sought. RESULTS: Six patients from a sample of 26 OCD images showed abnormalities at cluster or voxel level, considering the criteria described above, which represented 23.07%. However, seven images from the normal group of 32 were also indicated as cases of perfusional abnormality, representing 21.8% of the sample. CONCLUSION: The automated quantification method was not considered to be a useful tool for clinical practice, for analyses complementary to visual inspection.

Predictors of Atrial Fibrillation Recurrence in Hyperthyroid and Euthyroid Patients

Background: Atrial fibrillation (AF) is the most common arrhythmia in adults, and is encountered in 10-15% of the patients with hyperthyroidism. Unless euthyroidism is restored, pharmacological or electrical cardioversion is controversial in patients with AF who remain hyperthyroid. Objective: The aim of this study was to assess the efficacy of electrical cardioversion and predictors of AF recurrence in hyperthyroid and euthyroid patients. Methods: The study included 33 hyperthyroid (21 males) and 48 euthyroid (17 males) patients with persistent AF. The patients were sedated with intravenous midazolam before undergoing electrical cardioversion delivered by synchronized biphasic shocks. Rates of AF recurrence were recorded. Results: Mean follow-up was 23.63 ± 3.74 months in the hyperthyroid group and 22.78 ± 3.15 months in the euthyroid group (p = 0.51). AF recurred in 14 (43.8%) and 21 (44.7%) patients in each group, respectively (p = 0.93). Multivariate regression analysis in each group showed that AF duration was the only predictor of AF recurrence, with odds ratios of 1.38 (95% confidence interval [CI] = 1.05 - 1.82, p = 0.02) in the hyperthyroid group and 1.42 (95% CI = 1.05 - 1.91, p= 0.02) in the euthyroid group. Conclusion: Rates of long-term AF recurrence were similar in successfully cardioverted hyperthyroid and euthyroid patients. The only predictor of AF recurrence in both groups was AF duration.

Morphometric Analysis of McCoy Cells Inoculated with Cerebrospinal Fluid from Patients with Rabies

To demonstrate the potential of McCoy cells for the isolation of rabies virus from the cerebrospinal (CSF) fluid of a patient with a diagnosis of rabies, McCoy cells were inoculated with CSF from a patient with a clinical diagnosis of rabies and investigated in terms of morphometric aspect using the JAVA analysis system for the quantification of the increased size of infected cells compared to noninfected cells. The cells were also examined in terms of specific staining for the diagnosis of rabies by the method of Sellers for the observation of intracytoplasmic inclusions and by specific immunofluorescence staining for rabies virus. Infected cells showed changes in cell permeability and morphologic modifications which differed significantly compared to normal cells (P<0.001) when analyzed by the Mann-Whitney and Kruskal-Wallis tests. Intense activity of the endoplasmic reticulum was also observed, as indicated by the presence of intracytoplasmic inclusions visualized by specific staining. The present study demonstrated the isolation of rabies virus from the CSF of a patient with rabies, showing that McCoy cells can be used for the laboratory diagnosis of patients suspected to have rabies.

Real-time PCR-based quantification of Toxoplasma gondii in tissue samples of serologically positive outdoor chickens

This study aimed to quantify Toxoplasma gondii in tissue samples of serologically positive chickens using real-time polymerase chain reaction (PCR). Of 65 chickens evaluated, 28 were positive for T. gondii antibodies. Brain and heart samples were collected from 26 seropositive chickens and DNA was extracted using Trizol® and amplified using real-time PCR with SYBR® Green. Parasite DNA was detected in 24 of the 26 samples analyzed; the number of positive tissue samples and the parasite quantity did not differ between tissue types. The results confirmed the analytical sensitivity of parasite detection in chicken tissue samples and demonstrated the possibility of using other molecular systems for genotypic analysis.

Multivariate analysis and geostatistics of the fertility of a humic rhodic hapludox under coffee cultivation

The spatial variability of soil and plant properties exerts great influence on the yeld of agricultural crops. This study analyzed the spatial variability of the fertility of a Humic Rhodic Hapludox with Arabic coffee, using principal component analysis, cluster analysis and geostatistics in combination. The experiment was carried out in an area under Coffea arabica L., variety Catucai 20/15 - 479. The soil was sampled at a depth 0.20 m, at 50 points of a sampling grid. The following chemical properties were determined: P, K+, Ca2+, Mg2+, Na+, S, Al3+, pH, H + Al, SB, t, T, V, m, OM, Na saturation index (SSI), remaining phosphorus (P-rem), and micronutrients (Zn, Fe, Mn, Cu and B). The data were analyzed with descriptive statistics, followed by principal component and cluster analyses. Geostatistics were used to check and quantify the degree of spatial dependence of properties, represented by principal components. The principal component analysis allowed a dimensional reduction of the problem, providing interpretable components, with little information loss. Despite the characteristic information loss of principal component analysis, the combination of this technique with geostatistical analysis was efficient for the quantification and determination of the structure of spatial dependence of soil fertility. In general, the availability of soil mineral nutrients was low and the levels of acidity and exchangeable Al were high.

Quantification of the diversity among common bean accessions using Ward-MLM strategy

The present work aimed at evaluating the divergence among common bean accessions by their agronomic, morphological and molecular traits, based on the Ward-MLM procedure. A collection of 57 accessions from the gene bank of Universidade Federal do Espírito Santo was used in this study, from which: 31 were landraces belonging to the community Fortaleza, in the municipality of Muqui, ES, Brazil; 20 accessions were provided by Embrapa Trigo; and 6 were commercial cultivars. Five agronomic traits (plant cycle, number of seeds per pod, number of pods per plant, weight of 100 seeds, and grain yield), five morphological traits (growth habit, plant size, seed shape, seed color, and commercial group) and 16 microsatellite primers were evaluated. High genetic variability was detected considering morphological, agronomic and molecular traits in the 57 common bean accessions studied. The Ward-MLM procedure showed that the ideal number of groups was five, according to the pseudo F and pseudo t² criteria. The accessions from Andean origin had heavier seeds than others and formed a cluster. The Ward-MLM statistical procedure is a useful technique to detect genetic divergence and to cluster genotypes by simultaneously using morphological, agronomic and molecular data.

Relationship between two year PSA nadir and biochemical recurrence in prostate cancer patients treated with iodine-125 brachytherapy

Objective To evaluate the relationship between two year PSA nadir (PSAn) after brachytherapy and biochemical recurrence rates in prostate cancer patients. Materials and Methods In the period from January 1998 to August 2007, 120 patients were treated with iodine-125 brachytherapy alone. The results analysis was based on the definition of biochemical recurrence according to the Phoenix Consensus. Results Biochemical control was observed in 86 patients (71.7%), and biochemical recurrence, in 34 (28.3%). Mean PSAn was 0.53 ng/ml. The mean follow-up was 98 months. The patients were divided into two groups: group 1, with two year PSAn < 0.5 ng/ml after brachytherapy (74 patients; 61.7%), and group 2, with two year PSAn ≥ 0.5 ng/ml after brachytherapy (46 patients; 38.3%). Group 1 presented biochemical recurrence in 15 patients (20.3%), and group 2, in 19 patients (43.2%) (p < 0.02). The analysis of biochemical disease-free survival at seven years, stratified by the two groups, showed values of 80% and 64% (p < 0.02), respectively. Conclusion Levels of two year PSAn ≥ 0.5 ng/ml after brachytherapy are strongly correlated with a poor prognosis. This fact may help to identify patients at risk for disease recurrence.

Quantitative analysis of phenol and alkylphenols in Brazilian coal tar

The main purpose of this work is the identification and quantification of phenolic compounds in coal tar samples from a ceramics factory in Cocal (SC), Brazil. The samples were subjected to preparative scale liquid chromatography, using Amberlyst A-27TM ion-exchange resin as stationary phase. The fractions obtained were classified as "acids" and "BN" (bases and neutrals). The identification and quantification of phenols, in the acid fraction, was made by gas chromatography coupled to mass spectrometry (GC/MS). Nearly twenty-five phenols were identified in the samples and nine of them were also quantified. The results showed that coal tar has large quantities of phenolic compounds of industrial interest.