A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory.

111In-pentetreotide scintigraphy: procedure guidelines for tumour imaging.

This document provides general information about somatostatin receptor scintigraphy with (111)In-pentetreotide. This guideline should not be regarded as the only approach to visualise tumours expressing somatostatin receptors or as exclusive of other nuclear medicine procedures useful to obtain comparable results. The aim of this guideline is to assist nuclear medicine physicians in recommending, performing, reporting and interpreting the results of (111)In-pentetreotide scintigraphy.

TT virus infection in children and adults who visited a general hospital in the south of Brazil for routine procedure

TT virus (TTV) is a newly described nonenveloped human virus, with a circular, negative-stranded DNA genome, that was first identified in the blood of a patient with posttransfusion hepatitis of unknown etiology. PCR primers and conditions used for TTV DNA amplification may greatly influence the level of TTV detection in serum. Three PCR assays, with different regions of the genome as targets, were used to test TTV DNA in 130 sera from children and adults visiting a hospital in the south of Brazil, most of them for routine procedure. Forty-four percent of adult sera and 73% of sera from children aged 0-10 years were TTV positive with at least one PCR assay. However, the three assays were able to detect only 33%, 35%, and 70% of the total positive samples. Our results showed a high prevalence of TTV infection in the south of Brazil, particularly among young children, and confirmed the necessity of performing several PCR assays to assess the true TTV prevalence in a determined population.

Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidiniumthiocyanate DNA isolation procedure

With the objective to evaluate PCR-mediated detection of Mycobacterium tuberculosis DNA as a diagnostic procedure for diagnosis of tuberculosis in individuals attending ambulatory services in Primary Health Units of the City Tuberculosis Program in Rio de Janeiro, Brazil, their sputum samples were collected and treated with a DNA extraction procedure using silica-guanidiniumthiocyanate. This procedure has been described to be highly efficient for extraction of different kind of nucleic acids from bacteria and clinical samples. Upon comparing PCR results with the number of acid-fast bacilli, no direct relation was observed between the number of bacilli present in the sample and PCR positivity. Part of the processed samples was therefore spiked with pure DNA of M. tuberculosis and inhibition of the PCR reaction was verified in 22 out of 36 (61%) of the samples, demonstrating that the extraction procedure as originally described should not be used for PCR analysis of sputum samples.

Multiple nodules of the scrotum: histopathological findings and surgical procedure. A study of five cases.

BACKGROUND: Multiple nodules of the scrotum are uncommonly reported. Their origin is controversial. Treatment is always surgical but the best procedure is still to be determined. MATERIALS AND METHODS: Five new cases are reported with description of the histopathological findings and surgical procedure. RESULTS: Nodules of the scrotum were more frequent in patients with dark skin suggesting an ethnic susceptibility. No other predisposing factors were noted. Screening for disturbances of phosphate or calcium balance was negative. The following histopathological findings were observed: non-calcified epidermoid cysts (3 patients), calcified epidermoid cysts (1 patient) and nodular calcifications without epithelial or glandular structures (1 patient). Subtotal excisions of the scrotum wall using tumescent anaesthesia were performed in all patients without any significant complications. Cosmetic results were excellent. No new lesions were observed during the 1-year follow-up period. CONCLUSIONS: Most cases of multiple nodules of the scrotum are due to non-calcified epidermoid cysts. The term scrotal calcinosis is therefore probably abusively used by many authors. Some cases of nodular calcifications may be due to dystrophic calcification of epidermoid cysts, but calcifications may also occur without any visible epithelial or glandular structure. Subtotal excision of the scrotum wall is a safe and effective surgical procedure to treat multiple nodules of the scrotum. Cosmetic results are excellent and recurrences are rare.

Quantification of nicotine, cotinine, trans-3'-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation.

A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100mm, 1.7μm). A gradient program was used, with a 10mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Française des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500ng/mL for nicotine, 1-1000ng/mL for cotinine, 2-1000ng/mL for trans-3'-hydroxycotinine and 1-500ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.

Novel MLPA procedure using self-designed probes allows comprehensive analysis for CNVs of the genes involved in Hirschsprung disease.

Background: Hirschsprung disease is characterized by the absence of intramural ganglion cells in the enteric plexuses, due to a fail during enteric nervous system formation. Hirschsprung has a complex genetic aetiology and mutations in several genes have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. In this study, we have looked for CNVs in some of the genes related to Hirschsprung (EDNRB, GFRA1, NRTN and PHOX2B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach. Methods: CNVs screening was performed in 208 HSCR patients using a self-designed set of MLPA probes, covering the coding region of those genes. Results: A deletion comprising the first 4 exons in GFRA1 gene was detected in 2 sporadic HSCR patients and in silico approaches have shown that the critical translation initiation signal in the mutant gene was abolished. In this study, we have been able to validate the reliability of this technique for CNVs screening in HSCR. Conclusions: The implemented MLPA based technique presented here allows CNV analysis of genes involved in HSCR that have not been not previously evaluated. Our results indicate that CNVs could be implicated in the pathogenesis of HSCR, although they seem to be an uncommon molecular cause of HSCR.

Surgical procedure in immunoglobulin G4-related ascending aortitis?

Immunoglobulin G4 (IgG4)-related fibroinflammatory systemic disease accounts for 7% of all noninfectious aneurysms of the thoracic aorta. A patient was admitted with a symptomatic ascending aortic aneurysm and thickened aortic wall (outer/inner diameter 55/45 mm), which was replaced. Probes revealed IgG4-related aortitis associated with a primary tuberculosis infection. Corticosteroid and antituberculosis therapies were used, and the patient's clinical evolution was favorable. The optimal treatment strategy of IgG4-related aortitis, a new entity, remains vague. Inner aortic diameter alone does not justify aortic replacement, but wall thickening may mimic intramural hematoma. In this particular case of IgG4-related aortitis, immunosuppressive treatment alone, as an alternative to a surgical procedure, may be debatable.

Contrast ultrasound: a simple-to-use phase-shifting medium offers saline infusion sonography-like images.

OBJECTIVE: To test the ability of a novel phase-shifting medium (PSM) to provide sustained distension of the uterine cavity and produce saline infusion sonography (SIS)-like images in a simplified contrast ultrasound procedure. DESIGN: Prospective pilot feasibility trial of a new diagnostic procedure, contrast ultrasound. SETTING: Clinical reproductive endocrine and infertility unit of regional teaching hospital. PATIENT(S): Twenty-six asymptomatic infertile women (group I) and 27 women presenting with dysfunctional uterine bleeding (DUB) who were scheduled for exploratory surgery (group II). INTERVENTION(S): All women who were temporarily on oral contraceptive first had a regular pelvic ultrasound followed by the intrauterine instillation of up to 3 mL PSM, using a regular insemination catheter, after which all instruments were removed and a regular ultrasound was performed again. RESULT(S): In all 53 women, intrauterine instillation of 1-3 mL PSM resulted in a 3-7 mm uterine distension, sufficient to produce SIS-like images of the uterine cavity that lasted 7-10 min. Contrast ultrasound revealed an endometrial polyp in 3 asymptomatic women of group I. In group II. 12 of 14 women (86%) whose vaginal ultrasound were positive or dubious had positive findings with contrast ultrasound; 9 of 12 patients whose vaginal ultrasounds were negative also had positive contrast ultrasound findings. All the positive and negative findings of contrast ultrasound made in group II were confirmed anatomically (sensitivity and specificity of 100%), whereas the correlation for standard vaginal ultrasound was markedly lower at 57.1% and 85.7%, respectively. Most patients (46 of 53) reported no discomfort during or after the procedure, and 7 women described the procedure as mildly uncomfortable. CONCLUSION(S): Contrast ultrasound, a novel simple diagnostic procedure conducted after intrauterine instillation of 1-3 mL PSM using a simple plastic catheter, delivered SIS-quality images in asymptomatic (group I) and symptomatic (group II) patients while retaining the simplicity of standard ultrasound. We therefore foresee broad application of contrast ultrasound for sensitive and specific assessment for uterine pathologies in the physician's office.

Mise en place d'un cathéter veineux central chez l'adulte. 2. Technique détaillée [The central vein catheterization. 2. Procedure, step by step]

La pose d'un cathéter veineux central peut se faire par plusieurs abords. La connaissance de l'anatomie, du matériel, la maîtrise des détails de la procédure pour chaque type d'abord et une attention particulière à l'asepsie sont des prérequis importants pour limiter les risques de complications et pour donner une information complète et exhaustive au patient. Cet article peut être utilisé comme base pour le développement d'une check-list, utile pour les médecins qui doivent procéder à la pose d'un cathéter veineux central mais qui ne possèdent pas encore une maîtrise du geste. Several approaches exist for central vein catheterization. Mastery of the various steps of this procedure and understanding of the basics of asepsis are critical to prevent any complication. They also built the basis for an exhaustive communication with the patient, to obtain an informed consent. This article can in addition be used to develop a checklist in order to (self-)assess competence in procedural skills

An evaluation of the dot-ELISA procedure as a diagnostic test in an area with a high prevalence of human Toxocara canis infection

The aim of this work was to evaluate a dot-enzyme-linked immunosorbent assay (dot-ELISA) using excretory-secretory antigens from the larval stages of Toxocara canis for the diagnosis of toxocariasis. A secondary aim was to establish the optimal conditions for its use in an area with a high prevalence of human T. canis infection. The dot-ELISA test was standardised using different concentrations of the antigen fixed on nitrocellulose paper strips and increasing dilutions of the serum and conjugate. Both the dot-ELISA and standard ELISA methods were tested in parallel with the same batch of sera from controls and from individuals living in the problem area. The best results were obtained with 1.33 µg/mL of antigen, dilutions of 1/80 for the samples and controls and a dilution of 1/5,000 for the anti-human IgG-peroxidase conjugate. All steps of the procedure were performed at room temperature. The coincidence between ELISA and dot-ELISA was 85% and the kappa index was 0.72. The dot-ELISA test described here is rapid, easy to perform and does not require expensive equipment. Thus, this test is suitable for the serological diagnosis of human T. canis infection in field surveys and in the primary health care centres of endemic regions.

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

A new training set-up for trans-apical aortic valve replacement.

Trans-apical aortic valve replacement (AVR) is a new and rapidly growing therapy. However, there are only few training opportunities. The objective of our work is to build an appropriate artificial model of the heart that can replace the use of animals for surgical training in trans-apical AVR procedures. To reduce the necessity for fluoroscopy, we pursued the goal of building a translucent model of the heart that has nature-like dimensions. A simplified 3D model of a human heart with its aortic root was created in silico using the SolidWorks Computer-Aided Design (CAD) program. This heart model was printed using a rapid prototyping system developed by the Fab@Home project and dip-coated two times with dispersion silicone. The translucency of the heart model allows the perception of the deployment area of the valved-stent without using heavy imaging support. The final model was then placed in a human manikin for surgical training on trans-apical AVR procedure. Trans-apical AVR with all the necessary steps (puncture, wiring, catheterization, ballooning etc.) can be realized repeatedly in this setting.