Application of Real-Time PCR Stool Assay for Helicobacter pylori Detection and Clarithromycin Susceptibility Testing in Brazilian Children

Background: Helicobacter pylori ClariRes assay is a novel commercially available real-time PCR assay allowing H. pylori detection and clarithromycin susceptibility testing in either gastric biopsy or stool specimens. Objective: The aim of this study was to validate the novel biprobe real-time assay in stool specimens from 217 dyspeptic children. Methods: DNA from gastric biopsies and stool specimens were obtained and submitted to the biprobe real time assay for H. pylori detection and clarithromycin susceptibility testing. Results: The sensitivity, specificity, and test accuracy were 69, 100 and 93.9% for the detection of H. pylori infection and 83.3, 100 and 95.6%, for detection of clarithromycin resistance. Conclusion: This assay proved to be appropriate for H. pylori clarithromycin susceptibility testing, particularly in children populations where a high prevalence of clarithromycin-resistant strains is suspected.

Anemia During Pregnancy after Silastic Ring Roux-en-Y Gastric Bypass: Influence of Time to Conception

Bariatric surgery before pregnancy may help prevent obesity-related gestational complications. However, maternal malnutrition is not without potential risks during pregnancy. The objective was to evaluate the influence of time to conception after silastic ring Roux-en-Y gastric bypass (SRYGB) on maternal anemia. Patients who underwent SRYGB for morbid obesity and who subsequently became pregnant were followed up at the prenatal. Thirty pregnancies occurred between July 2001 and September 2009. The patients were analyzed according to time to conception after bariatric surgery: 17 patients with time to conception < 4 years (48 months) and 13 patients with a parts per thousand yen4 years. First trimester hemoglobin levels were significantly lower in patients with time to conception a parts per thousand yen4 years (48 months) (median 9.6 g/dL, range 5.8-13.2 g/dL) than in patients with time to conception < 4 years (median 11.1 g/dL, range 9.8-13.6 g/dL; p = 0.047). The need for intravenous iron therapy or packed red cell transfusion was significantly more frequent among women who became pregnant a parts per thousand yen4 years after SRYGB compared to < 4 years (30.8% vs. 0%, p = 0.026). Pregnancy after 4 years of SRYGB is associated with maternal anemia and the need for more strict iron supplementation.

miRNA analysis of prostate cancer by quantitative real time PCR: Comparison between formalin-fixed paraffin embedded and fresh-frozen tissue

Objective: Micro RNA (miRNA) is a class of small noncoding RNA that plays a major role in the regulation of gene expression, which has been related to cancer behavior. The possibility of analyzing miRNA from the archives of pathology laboratories is exciting, as it allows for large retrospective studies. Formalin is the most common fixative used in the surgical pathology routine, and its promotion of nucleic acid degradation is well known. Our aim is to compare miRNA profiles from formalin-fixed paraffin embedded (FFPE) tissues with fresh-frozen prostate cancer tissues. Methods: The expression of 14 miRNAs was determined by quantitative real time polymerase chain reaction (qRT-PCR) in 5 paired fresh-frozen and FFPE tissues, which were representative of prostate carcinoma. Results: There was a very good correlation of the miRNA expression of miR-let7c and miR-32 between the fresh-frozen and FFPE tissues, with Pearson`s correlation coefficients of 0.927 (P = 0.023) and 0.960 (P = 0.010), respectively. For the remaining miRNAs, the correlation was good with Spearman correlation coefficient of 0.638 (P < 0.001). Conclusion: Analysis of miRNAs from routinely processed and stored FFPE prostate tissue is feasible for some miRNAs using qRT-PCR. Further studies should be conducted to confirm the reliability of using stock tissues for miRNA expression determination. (C) 2011 Elsevier Inc. All rights reserved.

High-Resolution Genomic Mapping Reveals Consistent Amplification of the Fibroblast Growth Factor Receptor Substrate 2 Gene in Well-Differentiated and Dedifferentiated Liposarcoma

Well-differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31 CDK4, and others mainly derived from chromosome bands 12q13-15. However, the 12q13-15 amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high-resolution (5.0 kb) ""single nucleotide polymorphism""/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = 19) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho-FRS2 at 1436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating fibroblast growth factor receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma. (C) 2011 Wiley-Liss, Inc.

Pleural fluid: Are temperature and storage time critical preanalytical error factors in biochemical analyses?

Background: Biochemical analysis of fluid is the primary laboratory approach hi pleural effusion diagnosis. Standardization of the steps between collection and laboratorial analyses are fundamental to maintain the quality of the results. We evaluated the influence of temperature and storage time on sample stability. Methods: Pleural fluid from 30 patients was submitted to analyses of proteins, albumin, lactic dehydrogenase (LDH), cholesterol, triglycerides, and glucose. Aliquots were stored at 21 degrees, 4 degrees, and-20 degrees C, and concentrations were determined after 1, 2, 3, 4, 7, and 14 days. LDH isoenzymes were quantified in 7 random samples. Results: Due to the instability of isoenzymes 4 and 5, a decrease in LDH was observed in the first 24 h in samples maintained at -20 degrees C and after 2 days when maintained at 4 degrees C. Aside from glucose, all parameters were stable for up to at least day 4 when stored at room temperature or 4 degrees C. Conclusions: Temperature and storage time are potential preanalytical errors in pleural fluid analyses, mainly if we consider the instability of glucose and LDH. The ideal procedure is to execute all the tests immediately after collection. However, most of the tests can be done in refrigerated sample;, excepting LDH analysis. (C) 2010 Elsevier B.V. All rights reserved.

Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients

Cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients in Brazil, and results in high mortality and morbidity, despite free access to HAART (highly active antiretroviral treatment). Molecular diagnosis based on conventional PCR (cnPCR) or real-time quantitative PCR (qrtPCR) has been indispensable for definitive diagnosis. We report here the evaluation of qrtPCR with blood and cerebrospinal fluid (CSF) samples from AIDS patients in Brazil. This prospective study was conducted for 2 years, analysing DNA samples extracted from 149 AIDS patients (98 blood and 51 CSF samples) with confirmed clinical and radiological diagnosis The laboratory diagnosis included cnPCR (with the B22/B23 primer set) and indirect immunofluorescence (IF). For qrtPCR, two primer sets were simultaneously designed based on described genes and using a 6-carboxyfluorescein dye-labelled TaqMan MGB (minor groove binder) probe One was Bug, which amplified a sequence from the B1 gene The other was the RETg, which amplified a PCR product of the 529 bp sequence. The overall cnPCR and qrtPCR results were positive results were observed in 33.6% (50) patients The sensitivities were 98% for cnPCR (B22/B23), and 86 and 98% for qrtPCR (B1Tg and RETg, respectively). Negative reactions were observed in 66 4% patients. The specificities were 97% for cnPCR and qrtPCR (B1Tg). and 88.8% for RETg These data show that RETg PCR is highly sensitive as it amplifies a repeat region with many copies; however, its specificity is lower than the other markers However, B1Tg PCR had good specificity, but lower sensitivity Among the patients, 20 had blood and CSF collected simultaneously Thus, their results permitted us to analyse and compare molecular, serological and clinical diagnosis for a better understanding of the different scenarios of laboratorial and clinical diagnosis. For nine patients with confirmed cerebral toxoplasmosis diagnosis, four scenarios were observed: (i) and (ii) negative molecular diagnosis for CSF and positive for blood with variable IF titres for the sera and CSF (negative or positive), (iii) positive molecular diagnosis with CSF and negative with blood, and (iv) positive molecular diagnosis in both samples. In the latter two situations, normally the IF titres in sera and CSF are variable. Other opportunistic infections were shown in 11 patients Despite the IF titres in sera and CSF being variable, all of them had negative molecular diagnosis for both samples qrtPCR allows for a rapid identification of Toxoplasma gondii DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.

Time course of haemodynamic, respiratory and inflammatory disturbances induced by experimental acute pulmonary polystyrene microembolism

Background and objective The time course of cardiopulmonary alterations after pulmonary embolism has not been clearly demonstrated and nor has the role of systemic inflammation on the pathogenesis of the disease. This study aimed to evaluate over 12 h the effects of pulmonary embolism caused by polystyrene microspheres on the haemodynamics, lung mechanics and gas exchange and on interleukin-6 production. Methods Ten large white pigs (weight 35-42 kg) had arterial and pulmonary catheters inserted and pulmonary embolism was induced in five pigs by injection of polystyrene microspheres (diameter similar to 300 mu mol l(-1)) until a value of pulmonary mean arterial pressure of twice the baseline was obtained. Five other animals received only saline. Haemodynamic and respiratory data and pressure-volume curves of the respiratory system were collected. A bronchoscopy was performed before and 12 h after embolism, when the animals were euthanized. Results The embolism group developed hypoxaemia that was not corrected with high oxygen fractions, as well as higher values of dead space, airway resistance and lower respiratory compliance levels. Acute haemodynamic alterations included pulmonary arterial hypertension with preserved systemic arterial pressure and cardiac index. These derangements persisted until the end of the experiments. The plasma interleukin-6 concentrations were similar in both groups; however, an increase in core temperature and a nonsignificant higher concentration of bronchoalveolar lavage proteins were found in the embolism group. Conclusion Acute pulmonary embolism induced by polystyrene microspheres in pigs produces a 12-h lasting hypoxaemia and a high dead space associated with high airway resistance and low compliance. There were no plasma systemic markers of inflammation, but a higher central temperature and a trend towards higher bronchoalveolar lavage proteins were found. Eur J Anaesthesiol 27:67-76 (C) 2010 European Society of Anaesthesiology.

Obesity Decreases Time to Claudication and Delays Post-Exercise Hemodynamic Recovery in Elderly Peripheral Arterial Disease Patients

Background: Although obesity is usually observed in peripheral arterial disease (PAD) patients, the effects of the association between these diseases on walking capacity are not well documented. Objective: The main objectives of this study were to determine the effects of obesity on exercise tolerance and post-exercise hemodynamic recovery in elderly PAD patients. Methods: 46 patients with stable symptoms of intermittent claudication were classified according to their body mass index (BMI) into normal group (NOR) = BMI < 28.0 and obese or in risk of obesity group (OBE) = BMI >= 28.0. All patients performed a progressive graded treadmill test. During exercise, ventilatory responses were evaluated and pre- and post-exercise ankle and arm blood pressures were measured. Results: Exercise tolerance and oxygen consumption at total walking time were similar between OBE and NOR. However, OBE showed a lower claudication time (309 +/- 151 vs. 459 +/- 272 s, p = 0.02) with a similar oxygen consumption at this time. In addition, OBE presented a longer time for ankle brachial index recovery after exercise (7.8 +/- 2.8 vs. 6.3 +/- 2.6 min, p = 0.02). Conclusion: Obesity in elderly PAD patients decreased time to claudication, and delayed post-exercise hemodynamic recovery. These results suggest that muscle metabolic demand, and not total workload, is responsible for the start of the claudication and maximal exercise tolerance in PAD patients. Moreover, claudication duration might be responsible for the time needed to a complete hemodynamic recovery after exercise. Copyright (c) 2008 S. Karger AG, Basel