Evaluation of Adult Chronic Chagas` Heart Disease Diagnosis by Molecular and Serological Methods

Chagas` disease caused by Trypanosoma cruzi is endemic in Latin America. T. cruzi presents heterogeneous populations and comprises two main genetic lineages, named T. cruzi I and T. cruzi II. Diagnosis in the chronic phase is based on conventional serological tests, including indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA), and diagnosis in the acute phase based on parasitological methods, including hemoculture. The objective of this study was to evaluate the diagnostic procedures of Chagas` disease in adult patients in the chronic phase by using a PCR assay and conventional serological tests, including TESA-blot as the gold standard. Samples were obtained from 240 clinical chronic chagasic patients. The sensitivities, compared to that of TESA-blot, were 70% for PCR using the kinetoplast region, 75% for PCR using the nuclear repetitive region, 99% for IIF, and 95% for ELISA. According to the serological tests results, we recommend that researchers assess the reliability and sensitivity of the commercial kit Chagatest ELISA recombinant, version 3.0 (Chagatest Rec v3.0; Wiener Lab, Rosario, Argentina), due to the lack of sensitivity. Based on our analysis, we concluded that PCR cannot be validated as a conventional diagnostic technique for Chagas` disease. These data have been corroborated by low levels of concordance with serology test results. It is recommended that PCR be used only for alternative diagnostic support. Using the nuclear repetitive region of T. cruzi, PCR could also be applicable for monitoring patients receiving etiologic treatment.

Evaluation of computational methods for the reconstruction of HLA haplotypes

Human leukocyte antigen (HLA) haplotypes are frequently evaluated for population history inferences and association studies. However, the available typing techniques for the main HLA loci usually do not allow the determination of the allele phase and the constitution of a haplotype, which may be obtained by a very time-consuming and expensive family-based segregation study. Without the family-based study, computational inference by probabilistic models is necessary to obtain haplotypes. Several authors have used the expectation-maximization (EM) algorithm to determine HLA haplotypes, but high levels of erroneous inferences are expected because of the genetic distance among the main HLA loci and the presence of several recombination hotspots. In order to evaluate the efficiency of computational inference methods, 763 unrelated individuals stratified into three different datasets had their haplotypes manually defined in a family-based study of HLA-A, -B, -DRB1 and -DQB1 segregation, and these haplotypes were compared with the data obtained by the following three methods: the Expectation-Maximization (EM) and Excoffier-Laval-Balding (ELB) algorithms using the arlequin 3.11 software, and the PHASE method. When comparing the methods, we observed that all algorithms showed a poor performance for haplotype reconstruction with distant loci, estimating incorrect haplotypes for 38%-57% of the samples considering all algorithms and datasets. We suggest that computational haplotype inferences involving low-resolution HLA-A, HLA-B, HLA-DRB1 and HLA-DQB1 haplotypes should be considered with caution.

Clinical Evaluation of the NS1 Antigen-Capture ELISA for Early Diagnosis of Dengue Virus Infection in Brazil

The fact that the diagnosis of infection with dengue virus is usually made by detecting IgM antibodies during the convalescent phase of the disease interferes with disease management and, consequently, with reducing mortality rates. This study evaluated the sensitivity and specificity of detection of NS1 in samples of patients suspected of acute dengue virus infection in Brazil. The results were used to institute treatment and the sensitivity and specificity of detection of NS1 were compared to the results of detection of IgM, virus isolation, and RT-PCR. Detection of NS1 yielded better results than RTPCR and virus isolation. When considering IgM detection and RT-PCR positive results as ""gold standards,"" the sensitivity and specificity of the NS1 assay were 95.9% and 81.1%, respectively. All patients enrolled in the study were treated promptly and had an uneventful course of the disease. The detection of NS1 provided better results than the diagnostic techniques used currently during the acute phase of disease (RT-PCR and virus isolation). Detection of NS1 is an important tool for the diagnosis of acute dengue infection, particularly in highly endemic areas, allowing for rapid treatment of patients and reduction of disease burden. J. Med. Virol. 82: 1400-1405, 2010. (C) 2010 Wiley-Liss, Inc.

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: Myosin-v assay

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 degrees C, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.

THE INFLUENCE OF VIBRISSAL SOMATOSENSORY PROCESSING IN RAT SUPERIOR COLLICULUS ON PREY CAPTURE

The lateral part of intermediate layer of superior colliculus (SCI) is a critical substrate for successful predation by rats. Hunting-evoked expression of the activity marker Fos is concentrated in SCI while prey capture in rats with NMDA lesions in SCI is impaired. Particularly affected are rapid orienting and stereotyped sequences of actions associated with predation of fast moving prey. Such deficits are consistent with the view that the deep layers of SC are important for sensory guidance of movement. Although much of the relevant evidence involves visual control of movement, less is known about movement guidance by somatosensory input from vibrissae. Indeed, our impression is that prey contact with whiskers is a likely stimulus to trigger predation. Moreover, SCI receives whisker and orofacial somatosensory information directly from trigeminal complex, and indirectly from zona incerta, parvicelular reticular formation and somatosensory barrel cortex. To better understand sensory guidance of predation by vibrissal information we investigated prey capture by rats after whisker removal and the role of superior colliculus (SC) by comparing Fos expression after hunting with and without whiskers. Rats were allowed to hunt cockroaches, after which their whiskers were removed. Two days later they were allowed to hunt cockroaches again. Without whiskers the rats were less able to retain the cockroaches after capture and less able to pursue them in the event of the cockroach escaping. The predatory behaviour of rats with re-grown whiskers returned to normal. In parallel, Fos expression in SCI induced by predation was significantly reduced in whiskerless animals. We conclude that whiskers contribute to the efficiency of rat prey capture and that the loss of vibrissal input to SCI, as reflected by reduced Fos expression, could play a critical role in predatory deficits of whiskerless rats. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Methods for exploring the morpho-functional relations of the aortic depressor nerve in experimental diabetes

The present study investigated morpho-functional relations of the aortic depressor nerve (ADN) 5, 15 and 120 days after the onset of streptozotocin-induced diabetes in rats. Time control animals received vehicle. Under pentobarbital anesthesia, ADN activity was recorded simultaneously with arterial pressure. After the recordings, nerves were prepared for light microscopy study and morphometry. ADN function was accessed by means of pressure-nerve activity curve (fitted by sigmoidal regression) and cross-spectral analysis between mean arterial pressure (MAP) and ADN activity. The relation between morphological (myelinated fibers number and density, total myelin area, total fiber area and percentage of occupancy) and functional (gain, signal/noise relation, frequency) parameters were accessed by linear regression analysis and correlation coefficient calculations. Functional parameters obtained by means of the sigmoidal regression curve as well as by cross-spectral analysis were similar in diabetic and control rats. Morphometric parameters of the ADN were similar between groups 5 days after the onset of diabetes. Average myelin area and myelinated fiber area were significantly smaller on diabetic rats 15 and 120 days after the onset of diabetes, being the myelinated fiber and respective axons area and diameter also smaller on 120 days group. Nevertheless, G ratio (ratio between axon and fiber diameter) was nearly 0.6 and not different between groups or experimental times. No significant relationship between morphological and functional parameters was detected in all experimental groups. The present study suggests that ADN diabetic neuropathy was time-dependent, with damage to myelinated fibers to be the primary event, not evidenced by physiological methods. (C) 2010 Elsevier B.V. All rights reserved.

Evaluation of lignin contents in tropical forages using different analytical methods and their correlations with degradation of insoluble fiber

We compared the lignin contents of tropical forages by different analytical methods and evaluated their correlations with parameters related to the degradation of neutral detergent fiber (NDF). The lignin content was evaluated by five methods: cellulose solubilization in sulfuric acid [Lignin (sa)], oxidation with potassium permanganate [Lignin (pm)], the Klason lignin method (KL), solubilization in acetyl bromide from acid detergent fiber (ABLadf) and solubilization in acetyl bromide from the cell wall (ABLcw). Samples from ten grasses and ten legumes were used. The lignin content values obtained by gravimetric methods were also corrected for protein contamination, and the corrected values were referred to as Lignin (sa)p, Lignin (pm)p and KLp. The indigestible fraction of NDF (iNDF), the discrete lag (LAG) and the fractional rate of degradation (kd) of NDF were estimated using an in vitro assay. Correcting for protein resulted in reductions (P < 0.05) in the lignin contents as measured by the Lignin (sa), Lignin (pm) and, especially, the KL methods. There was an interaction (P < 0.05) of analytical method and forage group for lignin content. In general, LKp method provided the higher (P < 0.05) lignin contents. The estimates of lignin content obtained by the Lignin (sa)p, Lignin (pm)p and LKp methods were associated (P > 0.05) with all of the NDF degradation parameters. However, the strongest correlation coefficients for all methods evaluated were obtained with Lignin (pm)p and KLp. The lignin content estimated by the ABLcw method did not correlate (P > 0.05) with any parameters of NDF degradation. There was a correlation (P < 0.05) between the lignin content estimated by the ABLadf method and iNDF content. Nonetheless, this correlation was weaker than those found with gravimetric methods. From these results, we concluded that the gravimetric methods produce residues that are contaminated by nitrogenous compounds. Adjustment for these contaminants is suggested, particularly for the KL method, to express lignin content with greater accuracy. The relationships between lignin content measurements and NDF degradation parameters can be better determined using KLp and Lignin (pm)p methods. (C) 2011 Elsevier B.V. All rights reserved.