Droplet Digital PCR Detects Low-Density Infection in a Significant Proportion of Helicobacter Pylori-Negative Gastric Biopsies of Dyspeptic Patients.

INTRODUCTION: Helicobacter pylori-infected individuals may present low-density infection, undetectable by conventional tests such as histology, rapid urease test, or urea breath test. Droplet digital polymerase chain reaction (ddPCR) is more sensitive than other polymerase chain reaction methods. We aimed to evaluate the ability of ddPCR to detect H. pylori infection in patients diagnosed as negative by conventional tests. METHODS: Dyspeptic patients (n = 236) were tested for H. pylori by histology, urea breath test, and rapid urease test. Patients were classified as having 3 positive (n = 25, control group), 2 positive (n = 12), one positive (n = 41), or zero positive (n = 158) diagnostic tests. DNA was extracted from gastric biopsies. Triplicate ddPCR testing for each of the 16S rDNA, ureA, and vacA(s) genes was performed using a QX200 ddPCR system (Bio-Rad). A gene was considered positive when detected by at least 2 of 3 repeated ddPCRs. H. pylori positivity was defined as having 2 or more positive genes. RESULTS: All the biopsies of the control patients were positive for all 3 16S rDNA, ureA, and vacA(s) genes. H. pylori infection was detected in 57 (36%), 22 (54%), and 9 (75%) patients with zero, 1, and 2 positive diagnostic tests, respectively. The density of infection was 5, 121, 599, and 3,133 copies of H. pylori genome equivalents for patients with zero, 1, and 2 of 3 positive test results and for the control group, respectively. DISCUSSION: ddPCR detected low-density "occult" H. pylori infection in a significant proportion (36%) of patients diagnosed as negative by conventional methods. The number of conventional positive tests was related to the density of infection.

Hippocampal Damage During Mechanical Ventilation in Trendelenburg Position: A Secondary Analysis of an Experimental Study on the Prevention of Ventilator-Associated Pneumonia.

We previously corroborated benefits of the Trendelenburg position in the prevention of ventilator-associated pneumonia (VAP). We now investigate its potential effects on the brain versus the semirecumbent position. We studied 17 anesthetized pigs and randomized to be ventilated and positioned as follows: duty cycle (TI/TTOT) of 0.33, without positive end-expiratory pressure (PEEP), placed with the bed oriented 30° in anti-Trendelenburg (control group); positioned as in the control group, with TI/TTOT adjusted to achieve an expiratory flow bias, PEEP of 5 cm H2O (IRV-PEEP); positioned in 5° TP and ventilated as in the control group (TP). Animals were challenged into the oropharynx with Pseudomonas aeruginosa. We assessed hemodynamic parameters and systemic inflammation throughout the study. After 72 h, we evaluated incidence of microbiological/histological VAP and brain injury. Petechial hemorrhages score was greater in the TP group (P = 0.013). Analysis of the dentate gyrus showed higher cell apoptosis and deteriorating neurons in TP animals (P < 0.05 vs. the other groups). No differences in systemic inflammation were found among groups. Cerebral perfusion pressure was higher in TP animals (P < 0.001), mainly driven by higher mean arterial pressure. Microbiological/histological VAP developed in 0%, 67%, and 86% of the animals in the TP, control, and IRV-PEEP groups, respectively (P = 0.003). In conclusion, the TP prevents VAP; yet, we found deleterious neural effects in the dentate gyrus, likely associated with cerebrovascular modification in such position. Further laboratory and clinical studies are mandatory to appraise potential neurological risks associated with long-term TP.

Application of Individual qPCR Performance Parameters for Quality Control of Circulating MicroRNA Data.

Although circulating miRNAs are promising candidates for biomarkers, several challenges must be overcome before miRNAs can be used for diagnosis and monitoring. One is quality control for the RNA extraction and quantification process. RNA quality control techniques are unsuitable as circulating miRNAs are in the fM range. Additionally, biofluids may contain inhibitors of the reverse transcriptase and polymerase enzymes, which may survive RNA purification. Herein, we describe the protocol we have used to check the robustness of miRNA purification and measurement by the addition of spike-ins and by evaluating the quality of the qPCR data, respectively.

Usefulness of Housekeeping Genes for the Diagnosis of Helicobacter pylori Infection, Strain Discrimination and Detection of Multiple Infection.

BACKGROUND: Helicobacter pylori infects human stomachs of over half the world's population, evades the immune response and establishes a chronic infection. Although most people remains asymptomatic, duodenal and gastric ulcers, MALT lymphoma and progression to gastric cancer could be developed. Several virulence factors such as flagella, lipopolysaccharide, adhesins and especially the vacuolating cytotoxin VacA and the oncoprotein CagA have been described for H. pylori. Despite the extensive published data on H. pylori, more research is needed to determine new virulence markers, the exact mode of transmission or the role of multiple infection. MATERIALS AND METHODS: Amplification and sequencing of six housekeeping genes (amiA, cgt, cpn60, cpn70, dnaJ, and luxS) related to H. pylori pathogenesis have been performed in order to evaluate their usefulness for the specific detection of H. pylori, the genetic discrimination at strain level and the detection of multiple infection. A total of 52 H. pylori clones, isolated from 14 gastric biopsies from 11 patients, were analyzed for this purpose. RESULTS: All genes were specifically amplified for H. pylori and all clones isolated from different patients were discriminated, with gene distances ranged from 0.9 to 7.8%. Although most clones isolated from the same patient showed identical gene sequences, an event of multiple infection was detected in all the genes and microevolution events were showed for amiA and cpn60 genes. CONCLUSIONS: These results suggested that housekeeping genes could be useful for H. pylori detection and to elucidate the mode of transmission and the relevance of the multiple infection.

Good diagnostic accuracy of a chemiluminescent immunoassay in stool samples for diagnosis of Helicobacter pylori infection in patients with dyspepsia.

Laboratory-based chemiluminescence immunoassays (CLIA) are widely used in clinical laboratories. Some years ago, a CLIA test was developed for the detection of Helicobacter pylori in stool samples, known as LIAISON H. pylori SA, but little information on its use has been reported. To evaluate the accuracy of the LIAISON H. pylori SA assay for diagnosing H. pylori infection prior to eradication treatment. Diagnostic reliability was evaluated in 252 untreated consecutive patients with dyspepsia. The gold standard for diagnosing H. pylori infection was defined as the concordance of the rapid urease test (RUT), histopathology and urea breath test (UBT). The CLIA assay was performed according to the manufacturer's instructions. Sensitivity, specificity, positive and negative predictive values, and 95% CIs were calculated. According to the gold standard selected, 121 patients were positive for H. pylori infection and 131 negative. LIAISON H. pylori SA had a sensitivity of 90.1% and a specificity of 92.4%, with positive and negative predictive values of 91.6% and 90.1%, respectively. The accuracy of the LIAISON H. pylori SA chemiluminescent diagnostic assay seems comparable to that of ELISA or the best-performing LFIAs. Its sensitivity and specificity, however, seem slightly lower than those of histology, RUT or UBT. The advantages of the assay are that it is cheap, automated, and minimally labor-intensive.

Diagnostic accuracy of three monoclonal stool tests in a large series of untreated Helicobacter pylori infected patients.

OBJECTIVES: Immunochromatographic tests need to be improved in order to enhance their reliability. Recently, several new kits have appeared on the market. The objective was to evaluate the diagnostic accuracy of three monoclonal rapid stool tests - the new Uni-Gold™ H.pylori Antigen (Trinity Biotech, Ireland), the RAPID Hp StAR (Oxoid Ltd., UK) and the ImmunoCard STAT! HpSA (Meridian Diagnostics, USA) - for detecting H. pylori infection prior to eradication treatment. DESIGN AND METHODS: Diagnostic accuracy (sensitivity and specificity) and reliability (concordance between observers) were evaluated in 250 untreated consecutive dyspeptic patients. The gold standard for diagnosing H. pylori infection was defined as the concordance of two or more of rapid urease test (RUT), histopathology and urease breath test (UBT) or positive culture in isolation. Readings of immunochromatographic tests were performed by two different observers. Sensitivity, specificity, positive and negative predictive values and 95% confidence intervals were calculated. Sensitivity and specificity were compared using the McNemar test. RESULTS: The three tests showed a good correlation, with Kappa values>0.9. RAPID Hp StAR had a sensitivity of 91%-92% and a specificity ranging from 77% to 85%. Its sensitivity was higher than that of Uni-Gold™ H.pylori Antigen and ImmunoCard STAT! HpSA (p<0.01). Uni-Gold™ H.pylori Antigen kit showed a sensitivity of 83%, similar to ImmunoCard STAT! HpSA. Specificity of Uni-Gold™ H.pylori Antigen approached 90% (87-89%) and was superior to that of RAPID Hp StAR (p<0.01). CONCLUSIONS: Uni-Gold™ H.pylori Antigen and ImmunoCard STAT! HpSA present similar levels of diagnostic accuracy. RAPID Hp StAR was the most sensitive but less reliable of the three immunochromatographic stool tests. None are as accurate and reliable as UBT, RUT and histology.

Data on individual PCR efficiency values as quality control for circulating miRNAs.

This data article contains data related to the research article entitled "Variability in microRNA recovery from plasma: Comparison of five commercial kits, doi:10.1016/j.ab.2015.07.018" Brunet-Vega (2015) [1]. PCR efficiency, along with RNA and cDNA quality, are the most important factors affecting the quality of qPCR results. Constant amplification efficiency in all compared samples is indispensable when relative quantification is used to measure changes in gene expression. An easy way to measure PCR efficiency, without the need of a standard curve, is LinRegPCR software. Individual PCR efficiency can be determined as a part of qPCR quality control. This is especially important when the initial RNA quantity is so low that cannot be accurately quantified, such as in circulating RNA extractions. This data article reports the Cqs and PCR efficiencies of 5 miRNAs quantified in RNA isolated from 4 patients with colorectal cancer (CRC) and 4 healthy donors using five commercially available kits.

Moderate Peep After Tracheal Lipopolysaccharide Instillation Prevents Inflammation and Modifies the Pattern of Brain Neuronal Activation.

BACKGROUND: Ventilatory strategy and specifically positive end-expiratory pressure (PEEP) can modulate the inflammatory response and pulmonary-to-systemic translocation of lipopolysaccharide (LPS). Both inflammation and ventilatory pattern may modify brain activation, possibly worsening the patient's outcome and resulting in cognitive sequelae. METHODS: We prospectively studied Sprague-Dawley rats randomly assigned to undergo 3 h mechanical ventilation with 7 mL/kg tidal ventilation and either 2 cmH2O or 7 cmH2O PEEP after intratracheal instillation of LPS or saline. Healthy nonventilated rats served as baseline. We analyzed lung mechanics, gas exchange, lung and plasma cytokine levels, lung apoptotic cells, and lung neutrophil infiltration. To evaluate brain neuronal activation, we counted c-Fos immunopositive cells in the retrosplenial cortex (RS), thalamus, supraoptic nucleus (SON), nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), and central amygdala (CeA). RESULTS: LPS increased lung neutrophilic infiltration, lung and systemic MCP-1 levels, and neuronal activation in the CeA and NTS. LPS-instilled rats receiving 7 cmH2O PEEP had less lung and systemic inflammation and more c-Fos-immunopositive cells in the RS, SON, and thalamus than those receiving 2 cmH2O PEEP. Applying 7 cmH2O PEEP increased neuronal activation in the CeA and NTS in saline-instilled rats, but not in LPS-instilled rats. CONCLUSIONS: Moderate PEEP prevented lung and systemic inflammation secondary to intratracheal LPS instillation. PEEP also modified the neuronal activation pattern in the RS, SON, and thalamus. The relevance of these differential brain c-Fos expression patterns in neurocognitive outcomes should be explored.

Variability in microRNA recovery from plasma: Comparison of five commercial kits.

Numerous studies have indicated that microRNAs (miRNAs) are present and stable in multiple biological fluids, suggesting a great potential as biomarkers for molecular diagnostics and prognostics. Variations in the amount of starting material and isolation method to obtain miRNA may introduce bias and contribute to quantification errors. Given these concerns, we compared five commercially available kits for serum/plasma miRNA isolation to determine whether the plasma miRNA profile varies with the isolation method. We isolated miRNAs in blood plasma from colorectal cancer patients and healthy donors with five commercially available kits: Exiqon, Norgen, Macherey-Nagel, Qiagen, and Zymo Research. First, we assessed the robustness of the RNA isolation process and the quality of isolated miRNAs with the miRCURY microRNA QC PCR Panel (Exiqon), which contains six RNA spike-ins for quality control of RNA isolation (UniSp2, -4, and -5), complementary DNA (cDNA) synthesis (UniSp6 and cel-miR-39-3p), and polymerase chain reaction (PCR) amplification (UniSp3). This panel also includes circulating human miR-103, miR-191, miR-23a, and miR-451. Second, to evaluate the variability in miRNA profiling in relation to the extraction method, we analyzed plasma levels of candidate miRNA biomarkers for colorectal cancer (miR-18a, miR-21, and miR-29a). To determine PCR efficiencies per amplicon and per sample, we used LinRegPCR software. We found that all isolation methods were suitable for extracting miRNA from plasma samples and that all had similar Cq values in the three steps analyzed: RNA isolation, cDNA synthesis, and quantitative reverse transcription (qRT)-PCR. However, although the PCR replicates were excellent, the intersample variability of the spike-ins was unsatisfactorily high and all kits yielded suboptimal PCR efficiencies for some amplicons. Overall, our results underline the great difficulties involved in measuring miRNAs in plasma. The use of spike-ins is critical to control technical factors that affect final miRNA levels. We recommend that researchers investigating circulating miRNAs verify the PCR efficiency for each amplicon because quantification may be influenced by sample and PCR components.

Activation of the Wnt/ß-catenin signaling pathway by mechanical ventilation is associated with ventilator-induced pulmonary fibrosis in healthy lungs.

BACKGROUND: Mechanical ventilation (MV) with high tidal volumes (V(T)) can cause or aggravate lung damage, so-called ventilator induced lung injury (VILI). The relationship between specific mechanical events in the lung and the cellular responses that result in VILI remains incomplete. Since activation of Wnt/ß-catenin signaling has been suggested to be central to mechanisms of lung healing and fibrosis, we hypothesized that the Wnt/ß-catenin signaling plays a role during VILI. METHODOLOGY/PRINCIPAL FINDINGS: Prospective, randomized, controlled animal study using adult, healthy, male Sprague-Dawley rats. Animals (n = 6/group) were randomized to spontaneous breathing or two strategies of MV for 4 hours: low tidal volume (V(T)) (6 mL/kg) or high V(T) (20 mL/kg). Histological evaluation of lung tissue, measurements of WNT5A, total ß-catenin, non-phospho (Ser33/37/Thr41) ß-catenin, matrix metalloproteinase-7 (MMP-7), cyclin D1, vascular endothelial growth factor (VEGF), and axis inhibition protein 2 (AXIN2) protein levels by Western blot, and WNT5A, non-phospho (Ser33/37/Thr41) ß-catenin, MMP-7, and AXIN2 immunohistochemical localization in the lungs were analyzed. High-V(T) MV caused lung inflammation and perivascular edema with cellular infiltrates and collagen deposition. Protein levels of WNT5A, non-phospho (Ser33/37/Thr41) ß-catenin, MMP-7, cyclin D1, VEGF, and AXIN2 in the lungs were increased in all ventilated animals although high-V(T) MV was associated with significantly higher levels of WNT5A, non-phospho (Ser33/37/Thr41) ß-catenin, MMP-7, cyclin D1, VEGF, and AXIN2 levels. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that the Wnt/ß-catenin signaling pathway is modulated very early by MV in lungs without preexistent lung disease, suggesting that activation of this pathway could play an important role in both VILI and lung repair. Modulation of this pathway might represent a therapeutic option for prevention and/or management of VILI.

Injurious mechanical ventilation affects neuronal activation in ventilated rats.

INTRODUCTION: Survivors of critical illness often have significant long-term brain dysfunction, and routine clinical procedures like mechanical ventilation (MV) may affect long-term brain outcome. We aimed to investigate the effect of the increase of tidal volume (Vt) on brain activation in a rat model. METHODS: Male Sprague Dawley rats were randomized to three groups: 1) Basal: anesthetized unventilated animals, 2) low Vt (LVt): MV for three hours with Vt 8 ml/kg and zero positive end-expiratory pressure (ZEEP), and 3) high Vt (HVt) MV for three hours with Vt 30 ml/kg and ZEEP. We measured lung mechanics, mean arterial pressure (MAP), arterial blood gases, and plasma and lung levels of cytokines. We used immunohistochemistry to examine c-fos as a marker of neuronal activation. An additional group of spontaneously breathing rats was added to discriminate the effect of surgical procedure and anesthesia in the brain. RESULTS: After three hours on LVt, PaO2 decreased and PaCO2 increased significantly. MAP and compliance remained stable in MV groups. Systemic and pulmonary inflammation was higher in MV rats than in unventilated rats. Plasma TNFα was significantly higher in HVt than in LVt. Immunopositive cells to c-fos in the retrosplenial cortex and thalamus increased significantly in HVt rats but not in LVt or unventilated rats. CONCLUSIONS: MV promoted brain activation. The intensity of the response was higher in HVt animals, suggesting an iatrogenic effect of MV on the brain. These findings suggest that this novel cross-talking mechanism between the lung and the brain should be explored in patients undergoing MV.

Early physiological and biological features in three animal models of induced acute lung injury.

INTRODUCTION: Critically ill patients often develop acute lung injury (ALI) in the context of different clinical conditions. We aimed to explore differences in early local and systemic features in three experimental animal models of ALI. METHODS: Mechanically ventilated male Sprague-Dawley rats were randomized to high tidal volume (VT) ventilation (HVT) (n = 8, VT 24 ml/kg), massive brain injury (MBI) (n = 8, VT 8 ml/kg) or endotoxemia (LPS) (n = 8, VT 8 ml/kg). Each experimental group had its own control group of eight rats (VT 8 ml/kg). We measured arterial blood gases, mean arterial pressure, lung compliance, inflammatory mediators in plasma and their expression and gelatinase activity in the lungs after 3 h of injury. RESULTS: Despite maintaining relatively normal lung function without evidence of important structural changes, we observed altered lung and systemic inflammatory responses in all three experimental models. LPS triggered the most robust inflammatory response and HVT the lowest systemic proinflammatory response. The HVT group had higher Il6, Tnf and Cxcl2 mRNA in lungs than MBI animals. Metalloproteinase activity/expression and neutrophilic recruitment in the lungs were higher in HVT than in LPS or MBI. CONCLUSIONS: The early responses to direct or remote lung insult in our three models of ALI captured different physiological and biological features that could lead to respiratory and/or multiorgan failure.