Multicenter, Prospective Clinical Evaluation of Respiratory Samples from Subjects at Risk for Pneumocystis jirovecii Infection by Use of a Commercial Real-Time PCR Assay.

Pneumocystis jirovecii pneumonia (PCP) is a common opportunistic infection. Microscopic diagnosis, including diagnosis using the Merifluor-Pneumocystis direct fluorescent antigen (MP-DFA) test, has limitations. Real-time PCR may assist in diagnosis, but no commercially validated real-time PCR assay has been available to date. MycAssay Pneumocystis is a commercial assay that targets the P. jirovecii mitochondrial large subunit (analytical detection limit, ≤3.5 copies/μl of sample). A multicenter trial recruited 110 subjects: 54 with transplants (40 with lung transplants), 32 with nonmalignant conditions, 13 with leukemia, and 11 with solid tumors; 9 were HIV positive. A total of 110 respiratory samples (92% of which were bronchoalveolar lavage [BAL] specimens) were analyzed by PCR. Performance was characterized relative to investigator-determined clinical diagnosis of PCP (including local diagnostic tests), and PCR results were compared with MP-DFA test results for 83 subjects. Thirteen of 14 subjects with PCP and 9/96 without PCP (including 5 undergoing BAL surveillance after lung transplantation) had positive PCR results; sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) were 93%, 91%, 59%, and 99%, respectively. Fourteen of 83 subjects for whom PCR and MP-DFA test results were available had PCP; PCR sensitivity, specificity, PPV, and NPV were 93%, 90%, 65%, and 98%, respectively, and MP-DFA test sensitivity, specificity, PPV, and NPV were 93%, 100%, 100%, and 98%. Of the 9 PCR-positive subjects without PCP, 1 later developed PCP. The PCR diagnostic assay compares well with clinical diagnosis using nonmolecular methods. Additional positive results compared with the MP-DFA test may reflect low-level infection or colonization.

Application of real-time PCR for total airborne bacterial assessment : comparison with epifluorescence microscopy and culture-dependent methods

Traditional culture-dependent methods to quantify and identify airborne microorganisms are limited by factors such as short-duration sampling times and inability to count nonculturableor non-viable bacteria. Consequently, the quantitative assessment of bioaerosols is often underestimated. Use of the real-time quantitative polymerase chain reaction (Q-PCR) to quantify bacteria in environmental samples presents an alternative method, which should overcome this problem. The aim of this study was to evaluate the performance of a real-time Q-PCR assay as a simple and reliable way to quantify the airborne bacterial load within poultry houses and sewage treatment plants, in comparison with epifluorescencemicroscopy and culture-dependent methods. The estimates of bacterial load that we obtained from real-time PCR and epifluorescence methods, are comparable, however, our analysis of sewage treatment plants indicate these methods give values 270-290 fold greater than those obtained by the ''impaction on nutrient agar'' method. The culture-dependent method of air impaction on nutrient agar was also inadequate in poultry houses, as was the impinger-culture method, which gave a bacterial load estimate 32-fold lower than obtained by Q-PCR. Real-time quantitative PCR thus proves to be a reliable, discerning, and simple method that could be used to estimate airborne bacterial load in a broad variety of other environments expected to carry high numbers of airborne bacteria. [Authors]

Relationship between motion of coronary arteries and diaphragm during free breathing: lessons from real-time MR imaging.

OBJECTIVE: Diaphragmatic navigators are frequently used in free-breathing coronary MR angiography, either to gate or prospectively correct slice position or both. For such approaches, a constant relationship between coronary and diaphragmatic displacement throughout the respiratory cycle is assumed. The purpose of this study was to evaluate the relationship between diaphragmatic and coronary artery motion during free breathing. SUBJECTS AND METHODS: A real-time echoplanar MR imaging sequence was used in 12 healthy volunteers to obtain 30 successive images each (one per cardiac cycle) that included the left main coronary artery and the domes of both hemidiaphragms. The coronary artery and diaphragm positions (relative to isocenter) were determined and analyzed for effective diaphragmatic gating windows of 3, 5, and 7 mm (diaphragmatic excursions of 0-3, 0-5, and 0-7 mm from the end-expiratory position, respectively). RESULTS: Although the mean slope correlating the displacement of the right diaphragm and the left main coronary artery was approximately 0.6 for all diaphragmatic gating windows, we also found great variability among individual volunteers. Linear regression slopes varied from 0.17 to 0.93, and r2 values varied from .04 to .87. CONCLUSION: Wide individual variability exists in the relationship between coronary and diaphragmatic respiratory motion during free breathing. Accordingly, coronary MR angiographic approaches that use diaphragmatic navigator position for prospective slice correction may benefit from patient-specific correction factors. Alternatively, coronary MR angiography may benefit from a more direct assessment of the respiratory displacement of the heart and coronary arteries, using left ventricular navigators.

Analog circuit for real-time computation of respiratory mechanical impedance in sleep studies

The aim of this work was to develop a low-cost circuit for real-time analog computation of the respiratory mechanical impedance in sleep studies. The practical performance of the circuit was tested in six patients with obstructive sleep apnea. The impedance signal provided by the analog circuit was compared with the impedance calculated simultaneously with a conventional computerized system. We concluded that the low-cost analog circuit developed could be a useful tool for facilitating the real-time assessment of airway obstruction in routine sleep studies.

Submillimeter three-dimensional coronary MR angiography with real-time navigator correction: comparison of navigator locations.

Three-dimensional free-breathing coronary magnetic resonance angiography was performed in eight healthy volunteers with use of real-time navigator technology. Images acquired with the navigator localized at the right hemidiaphragm and at the left ventricle were objectively compared. The diaphragmatic navigator was found to be superior for vessel delineation of middle to distal portions of the coronary arteries.

Linking Real Time and Location in Scheduling Demand-Responsive Transit, September 1996

The role of rural demand-responsive transit is changing, and with that change is coming an increasing need for technology. As long as rural transit was limited to a type of social service transportation for a specific set of clients who primarily traveled in groups to common meal sites, work centers for the disabled, or clinics in larger communities, a preset calendar augmented by notes on a yellow legal pad was sufficient to develop schedules. Any individual trips were arranged at least 24 to 48 hours ahead of time and were carefully scheduled the night before in half-hour or twenty-minute windows by a dispatcher who knew every lane in the service area. Since it took hours to build the schedule, any last-minute changes could wreak havoc with the plans and raise the stress level in the dispatch office. Nevertheless, given these parameters, a manual scheduling system worked for a small demand-responsive operation.

Real-time motion correction in navigator-gated free-breathing double-oblique submillimeter 3D right coronary artery magnetic resonance angiography.

RATIONALE AND OBJECTIVES: The purpose of this study was the investigation of the impact of real-time adaptive motion correction on image quality in navigator-gated, free-breathing, double-oblique three-dimensional (3D) submillimeter right coronary magnetic resonance angiography (MRA). MATERIALS AND METHODS: Free-breathing 3D right coronary MRA with real-time navigator technology was performed in 10 healthy adult subjects with an in-plane spatial resolution of 700 x 700 microm. Identical double-oblique coronary MR-angiograms were performed with navigator gating alone and combined navigator gating and real-time adaptive motion correction. Quantitative objective parameters of contrast-to-noise ratio (CNR) and vessel sharpness and subjective image quality scores were compared. RESULTS: Superior vessel sharpness, increased CNR, and superior image quality scores were found with combined navigator gating and real-time adaptive motion correction (vs. navigator gating alone; P < 0.01 for all comparisons). CONCLUSION: Real-time adaptive motion correction objectively and subjectively improves image quality in 3D navigator-gated free-breathing double-oblique submillimeter right coronary MRA.

Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of rRNA.

Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either the 16S rRNA genes or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost > or =4 log(10) CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost < or =1 log(10) CFU/ml. Amplification of a 427-bp fragment of 16S rRNA genes yielded amplicons that increased proportionally to viable counts during bacterial growth but did not decrease during drug-induced killing. In contrast, the same 427-bp fragment amplified from 16S rRNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Tol1 mutant (> or =4 log(10) CFU/ml and < or =1 log(10) CFU/ml, respectively) and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments, the experiments were repeated by amplifying a 119-bp region internal to the original 427-bp fragment. The amount of 119-bp amplicons increased proportionally to viability during growth but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical for differentiation between live and dead bacteria.

Analog circuit for real-time computation of respiratory mechanical impedance in sleep studies

The aim of this work was to develop a low-cost circuit for real-time analog computation of the respiratory mechanical impedance in sleep studies. The practical performance of the circuit was tested in six patients with obstructive sleep apnea. The impedance signal provided by the analog circuit was compared with the impedance calculated simultaneously with a conventional computerized system. We concluded that the low-cost analog circuit developed could be a useful tool for facilitating the real-time assessment of airway obstruction in routine sleep studies.

Viscoelasticity in Achilles Tendonopathy: Quantitative Assessment by Using Real-time Shear-Wave Elastography.

Purpose To investigate the differences in viscoelastic properties between normal and pathologic Achilles tendons ( AT Achilles tendon s) by using real-time shear-wave elastography ( SWE shear-wave elastography ). Materials and Methods The institutional review board approved this study, and written informed consent was obtained from 25 symptomatic patients and 80 volunteers. One hundred eighty ultrasonographic (US) and SWE shear-wave elastography studies of AT Achilles tendon s without tendonopathy and 30 studies of the middle portion of the AT Achilles tendon in patients with tendonopathy were assessed prospectively. Each study included data sets acquired at B-mode US (tendon morphology and cross-sectional area) and SWE shear-wave elastography (axial and sagittal mean velocity and relative anisotropic coefficient) for two passively mobilized ankle positions. The presence of AT Achilles tendon tears at B-mode US and signal-void areas at SWE shear-wave elastography were noted. Results Significantly lower mean velocity was shown in tendons with tendonopathy than in normal tendons in the relaxed position at axial SWE shear-wave elastography (P < .001) and in the stretched position at sagittal (P < .001) and axial (P = .0026) SWE shear-wave elastography . Tendon softening was a sign of tendonopathy in relaxed AT Achilles tendon s when the mean velocity was less than or equal to 4.06 m · sec(-1) at axial SWE shear-wave elastography (sensitivity, 54.2%; 95% confidence interval [ CI confidence interval ]: 32.8, 74.4; specificity, 91.5%; 95% CI confidence interval : 86.3, 95.1) and less than or equal to 5.70 m · sec(-1) at sagittal SWE shear-wave elastography (sensitivity, 41.7%; 95% CI confidence interval : 22.1, 63.3; specificity, 81.8%; 95% CI confidence interval : 75.3, 87.2) and in stretched AT Achilles tendon s, when the mean velocity was less than or equal to 4.86 m · sec(-1) at axial SWE shear-wave elastography (sensitivity, 66.7%; 95% CI confidence interval : 44.7, 84.3; specificity, 75.6%; 95% CI confidence interval : 68.5, 81.7) and less than or equal to 14.58 m · sec(-1) at sagittal SWE shear-wave elastography (sensitivity, 58.3%; 95% CI confidence interval : 36.7, 77.9; specificity, 83.5%; 95% CI confidence interval : 77.2, 88.7). Anisotropic results were not significantly different between normal and pathologic AT Achilles tendon s. Six of six (100%) partial-thickness tears appeared as signal-void areas at SWE shear-wave elastography . Conclusion Whether the AT Achilles tendon was relaxed or stretched, SWE shear-wave elastography helped to confirm and quantify pathologic tendon softening in patients with tendonopathy in the midportion of the AT Achilles tendon and did not reveal modifications of viscoelastic anisotropy in the tendon. Tendon softening assessed by using SWE shear-wave elastography appeared to be highly specific, but sensitivity was relatively low. © RSNA, 2014.

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor. Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

Using Scanning Lasers for Real-Time Pavement Thickness Measurement, TR-538, 2006

Due to limited budgets and reduced inspection staff, state departments of transportation (DOTs) are in need of innovative approaches for providing more efficient quality assurance on concrete paving projects. The goal of this research was to investigate and test new methods that can determine pavement thickness in real time. Three methods were evaluated: laser scanning, ultrasonic sensors, and eddy current sensors. Laser scanning, which scans the surface of the base prior to paving and then scans the surface after paving, can determine the thickness at any point. Also, scanning lasers provide thorough data coverage that can be used to calculate thickness variance accurately and identify any areas where the thickness is below tolerance. Ultrasonic and eddy current sensors also have the potential to measure thickness nondestructively at discrete points and may result in an easier method of obtaining thickness. There appear to be two viable approaches for measuring concrete pavement thickness during the paving operation: laser scanning and eddy current sensors. Laser scanning has proved to be a reliable technique in terms of its ability to provide virtual core thickness with low variability. Research is still required to develop a prototype system that integrates point cloud data from two scanners. Eddy current sensors have also proved to be a suitable alternative, and are probably closer to field implementation than the laser scanning approach. As a next step for this research project, it is suggested that a pavement thickness measuring device using eddy current sensors be created, which would involve both a handheld and paver-mounted version of the device.

Exposure to bioaerosols in poultry houses at different stages of fattening; use of real-time PCR for airborne bacterial quantification

Previous studies have demonstrated that poultry house workers are exposed to very high levels of organic dust and consequently have an increased prevalence of adverse respiratory symptoms. However, the influence of the age of broilers on bioaerosol concentrations has not been investigated. To evaluate the evolution of bioaerosol concentration during the fattening period, bioaerosol parameters (inhalable dust, endotoxin and bacteria) were measured in 12 poultry confinement buildings in Switzerland, at three different stages of the birds' growth; samples of air taken from within the breathing zones of individual poultry house employees as they caught the chickens ready to be transported for slaughter were also analysed. Quantitative polymerase chain reaction (Q-PCR) was used to assess the quantity of total airborne bacteria and total airborne Staphylococcus species. Bioaerosol levels increased significantly during the fattening period of the chickens. During the task of catching mature birds, the mean inhalable dust concentration for a worker was 26 +/- 1.9 mg m(-3) and endotoxin concentration was 6198 +/- 2.3 EU m(-3) air, >6-fold higher than the Swiss occupational recommended value (1000 EU m(-3)). The mean exposure level of bird catchers to total bacteria and Staphylococcus species measured by Q-PCR is also very high, respectively, reaching values of 53 (+/-2.6) x 10(7) cells m(-3) air and 62 (+/-1.9) x 10(6) m(-3) air. It was concluded that in the absence of wearing protective breathing apparatus, chicken catchers in Switzerland risk exposure beyond recommended limits for all measured bioaerosol parameters. Moreover, the use of Q-PCR to estimate total and specific numbers of airborne bacteria is a promising tool for evaluating any modifications intended to improve the safety of current working practices