Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis

Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3 h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres.

Ireland after Rapid Immigration:The consequences of neglect

Essay review of:
Immigration and Schooling in the Republic of Ireland: Making a Difference?, by Dympna Devine . Manchester, UK: Manchester University Press, 2012. 186pp. $24.95 paper. ISBN: 9780719081026.
Immigration and Social Cohesion in the Republic of Ireland, by Bryan Fanning . Manchester, UK: Manchester University Press, 2011. 202pp. $24.95 paper. ISBN: 9780719084799.
Understanding Immigration in Ireland: State, Capital and Labour in a Global Age, by Steven Loyal . Manchester, UK: Manchester University Press, 2011. 283pp. $24.95 paper. ISBN: 9780719078316.

Multiplex Touchdown PCR for Rapid Typing of the Opportunistic Pathogen Propionibacterium acnes.

The opportunistic human pathogen Propionibacterium acnes is comprised of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II and III, that vary in their production of putative virulence factors, inflammatory potential, as well as biochemical, aggregative and morphological characteristics. Although Multilocus Sequence Typing (MLST) currently represents the gold standard for unambiguous phylogroup classification, and individual strain identification, it is a labour and time-consuming technique. As a consequence, we have developed a multiplex touchdown PCR assay that will, in a single reaction, confirm species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA (all isolates), ATPase (type IA1, IA2, IC), sodA (type IA2, IB), atpD (type II) and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). When applied to 312 P. acnes isolates previously characterised by MLST, and representing type IA1 (n=145), IA2 (n=20), IB (n=65), IC (n=7), II (n=45) and III (n=30), the multiplex displayed 100% sensitivity and 100% specificity for the detection of isolates within each targeted phylogroup. No cross-reactivity with isolates from other bacterial species was observed. The multiplex assay will provide researchers with a rapid, high-throughput and technically undemanding typing method for epidemiological and phylogenetic investigations. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, as well as a pre-screening tool to maximise the number of genetically diverse isolates selected for downstream, higher resolution sequence-based analyses.

Development of a rapid multiplexed assay for the direct screening of antimicrobial residues in raw milk

Antimicrobial residues found to be present in milk can have both health and economic impacts. For these reasons, the widespread routine testing of milk is required. Due to delays with sample handling and test scheduling, laboratory-based tests are not always suited for making decisions about raw material intake and product release, especially when samples require shipping to a central testing facility. Therefore, rapid on-site screening tests that can produce results within a matter of minutes are required to facilitate rapid intake and product release processes. Such tests must be simple for use by non-technical staff. There is increasing momentum towards the development and implementation of multiplexing tests that can detect a range of important antimicrobial residues simultaneously. A simple in situ multiplexed planar waveguide device that can simultaneously detect chloramphenicol, streptomycin and desfuroylceftiofur in raw dairy milk, without sample preparation, has been developed. Samples are simply mixed with antibody prior to an aliquot being passed through the detection cartridge for 5 min before reading on a field-deployable portable instrument. Multiplexed calibration curves were produced in both buffer and raw milk. Buffer curves, for chloramphenicol, streptomycin and desfuroylceftiofur, showed linear ranges (inhibitory concentration (IC)₂₀–IC₈₀) of 0.1–0.9, 3–129 and 12–26 ng/ml, whilst linear range in milk was 0.13–0.74, 11–376 and 2–12 ng/ml, respectively, thus meeting European legislated concentration requirements for both chloramphenicol and streptomycin, in milk, without the need for any sample preparation. Desfuroylceftiofur-contaminated samples require only simple sample dilution to bring positive samples within the range of quantification. Assay repeatability and reproducibility were lower than 12 coefficient of variation (%CV), whilst blank raw milk samples (n = 9) showed repeatability ranging between 4.2 and 8.1 %CV when measured on all three calibration curves.

'Leptorapide' - a one-step assay for rapid diagnosis of human leptospirosis

Leptospirosis is a globally important zoonotic infection caused by spirochaetes of the genus Leptospira. It is transmitted to humans by direct contact with infected animals or indirectly via contaminated water. It is mainly a problem of the resource-poor developing countries of the tropical and sub-tropical regions of the world but outbreaks due to an increase in travel and recreational activities have been reported in developed and more industrialized areas of the world. Current methods of diagnosis are costly, time-consuming and require the use of specialized laboratory equipment and personnel. The purpose of this paper is to report the validation of the 'Leptorapide®' test (Linnodee Ltd, Northern Ireland) for the diagnosis of human leptospirosis. It is a simple one-step latex agglutination assay performed using equal volumes of serum sample and antigen-bound latex beads. Evidence of leptospiral antibodies is determined within minutes. Agglutination is scored on a scale of 1-5 and the results interpreted using a score card provided with the kit. Validation has been performed with a large sample size obtained from individuals originating from various parts of the world including Brazil and India. The test has shown sensitivity and specificity values of 97·1% and 94·0%, respectively, relative to the microscopic agglutination test. The results demonstrate that Leptorapide offers a cost-effective and accurate alternative to the more historical methods of antibody detection.

Rapid quantification of microRNAs in plasma using a fast real time PCR system

The ability to rapidly detect circulating small RNAs, in particular microRNAs (miRNAs), would further increase their already established potential as biomarkers in a range of conditions. One rate-limiting factor is the time taken to perform quantitative real time PCR amplification. We therefore evaluated the ability of a novel thermal cycler to perform this step in less than 10 minutes. Quantitative PCR was performed on an xxpress® thermal cycler (BJS Biotechnologies, Perivale, UK), which employs a resistive heating system and forced air cooling to achieve thermal ramp rates of 10 °C/s, and a conventional peltier-controlled LightCycler 480 system (Roche, Basel, Switzerland) ramping at 4.8 °C/s. The threshold cycle (Ct) for detection of 18S rDNA from a standard genomic DNA sample was significantly more variable across the block (F-test, p=2.4x10-25) for the xxpress (20.01±0.47SD) than the LightCycler (19.87±0.04SD). RNA was extracted from human plasma, reverse transcribed and a panel of miRNAs amplified and detected using SYBR green (Kapa Biosystems, Wilmington, Ma, USA). The sensitivity of both systems was broadly comparable and both detected a panel of miRNAs reliably and indicated similar relative abundances. The xxpress thermal cycler facilitates rapid qPCR detection of small RNAs and brings point-of care diagnostics based upon circulating miRNAs a step closer to reality.

Rapid detection of health-care-associated bloodstream infection in critical care using multipathogen real-time polymerase chain reaction technology: a diagnostic accuracy study and systematic review

Background: There is growing interest in the potential utility of real-time polymerase chain reaction (PCR) in diagnosing bloodstream infection by detecting pathogen deoxyribonucleic acid (DNA) in blood samples within a few hours. SeptiFast (Roche Diagnostics GmBH, Mannheim, Germany) is a multipathogen probe-based system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection. As background to this study, we report a systematic review of Phase III diagnostic accuracy studies of SeptiFast, which reveals uncertainty about its likely clinical utility based on widespread evidence of deficiencies in study design and reporting with a high risk of bias. 

Objective: Determine the accuracy of SeptiFast real-time PCR for the detection of health-care-associated bloodstream infection, against standard microbiological culture. 

Design: Prospective multicentre Phase III clinical diagnostic accuracy study using the standards for the reporting of diagnostic accuracy studies criteria. 

Setting: Critical care departments within NHS hospitals in the north-west of England. 

Participants: Adult patients requiring blood culture (BC) when developing new signs of systemic inflammation. 

Main outcome measures: SeptiFast real-time PCR results at species/genus level compared with microbiological culture in association with independent adjudication of infection. Metrics of diagnostic accuracy were derived including sensitivity, specificity, likelihood ratios and predictive values, with their 95% confidence intervals (CIs). Latent class analysis was used to explore the diagnostic performance of culture as a reference standard. 

Results: Of 1006 new patient episodes of systemic inflammation in 853 patients, 922 (92%) met the inclusion criteria and provided sufficient information for analysis. Index test assay failure occurred on 69 (7%) occasions. Adult patients had been exposed to a median of 8 days (interquartile range 4–16 days) of hospital care, had high levels of organ support activities and recent antibiotic exposure. SeptiFast real-time PCR, when compared with culture-proven bloodstream infection at species/genus level, had better specificity (85.8%, 95% CI 83.3% to 88.1%) than sensitivity (50%, 95% CI 39.1% to 60.8%). When compared with pooled diagnostic metrics derived from our systematic review, our clinical study revealed lower test accuracy of SeptiFast real-time PCR, mainly as a result of low diagnostic sensitivity. There was a low prevalence of BC-proven pathogens in these patients (9.2%, 95% CI 7.4% to 11.2%) such that the post-test probabilities of both a positive (26.3%, 95% CI 19.8% to 33.7%) and a negative SeptiFast test (5.6%, 95% CI 4.1% to 7.4%) indicate the potential limitations of this technology in the diagnosis of bloodstream infection. However, latent class analysis indicates that BC has a low sensitivity, questioning its relevance as a reference test in this setting. Using this analysis approach, the sensitivity of the SeptiFast test was low but also appeared significantly better than BC. Blood samples identified as positive by either culture or SeptiFast real-time PCR were associated with a high probability (> 95%) of infection, indicating higher diagnostic rule-in utility than was apparent using conventional analyses of diagnostic accuracy. 

Conclusion: SeptiFast real-time PCR on blood samples may have rapid rule-in utility for the diagnosis of health-care-associated bloodstream infection but the lack of sensitivity is a significant limiting factor. Innovations aimed at improved diagnostic sensitivity of real-time PCR in this setting are urgently required. Future work recommendations include technology developments to improve the efficiency of pathogen DNA extraction and the capacity to detect a much broader range of pathogens and drug resistance genes and the application of new statistical approaches able to more reliably assess test performance in situation where the reference standard (e.g. blood culture in the setting of high antimicrobial use) is prone to error.

The Dynamics of Rapid Redshifted and Blueshifted Excursions in the Solar Hα Line

We analyze high temporal and spatial resolution time-series of spectralscans of the Hα line obtained with the CRisp Imaging SpectroPolarimeter instrument mounted on the Swedish Solar Telescope.The data reveal highly dynamic, dark, short-lived structures known asRapid Redshifted and Blueshifted Excursions (RREs, RBEs) that areon-disk absorption features observed in the red and blue wings ofspectral lines formed in the chromosphere. We study the dynamics of RREsand RBEs by tracking their evolution in space and time, measuring thespeed of the apparent motion, line of sight (LOS) Doppler velocity, andtransverse velocity of individual structures. A statistical study oftheir measured properties shows that RREs and RBEs have similaroccurrence rates, lifetimes, lengths, and widths. They also displaynon-periodic, nonlinear transverse motions perpendicular to their axesat speeds of 4-31 km s-1. Furthermore, both typesof structures either appear as high speed jets and blobs that aredirected outwardly from a magnetic bright point with speeds of50-150 km s-1, or emerge within a few seconds. Astudy of the different velocity components suggests that the transversemotions along the LOS of the chromospheric flux tubes are responsiblefor the formation and appearance of these redshifted/blueshiftedstructures. The short lifetime and fast disappearance of the RREs/RBEssuggests that, similar to type II spicules, they are rapidly heated totransition region or even coronal temperatures. We speculate that theKelvin-Helmholtz instability triggered by observed transversemotions of these structures may be a viable mechanism for their heating.

Oxidation of N-(4-methoxybenzyl)-2-pyrrolidinones to N-(4-methoxybenzoyl)-2-pyrrolidinones. Rapid entry to optically active Aniracetam analogues.

Oxidation of readily available N-(4-methoxybenzyl)-5-alkylpyrrolidin-2-ones to the corresponding N-(4-methoxybenzoyl)-5-alkylpyrrolidin-2-ones gives direct access to enantiomerically pure 5-alkyl analogues of the cognition activating agent Aniracetam. Copyright (C) 1996 Elsevier Science Ltd

Turbulent Fluctuations in G-band and K-line Intensities Observed with the Rapid Oscillations in the Solar Atmosphere (ROSA) Instrument

Using the Rapid Oscillation in the Solar Atmosphere (ROSA) instrument at the Dunn Solar Telescope we have found that the spectra of fluctuations of the G-band (cadence 1.05 s) and Ca II K-line (cadence 4.2 s) intensities show correlated fluctuations above white noise out to frequencies beyond 300 mHz and up to 70 mHz, respectively. The noise-corrected G-band spectrum presents a scaling range (Ultra High Frequency “UHF”) for f = 25-100 mHz, with an exponent consistent with the presence of turbulent motions. The UHF power, is concentrated at the locations of magnetic bright points in the intergranular lanes, it is highly intermittent in time and characterized by a positive kurtosis κ. Combining values of G-band and K-line intensities, the UHF power, and κ, reveals two distinct “states” of the internetwork solar atmosphere. State 1, with κ ≍ 6, which includes almost all the data, is characterized by low intensities and low UHF power. State 2, with κ ≍ 3, including a very small fraction of the data, is characterized by high intensities and high UHF power. Superposed epoch analysis shows that for State 1, the K-line intensity presents 3.5 min chromospheric oscillations with maxima occurring 21 s after G-band intensity maxima implying a 150-210 km effective height difference. For State 2, the G-band and K-line intensity maxima are simultaneous, suggesting that in the highly magnetized environment sites of G-band and K-line emission may be spatially close together. Analysis of observations obtained with Hinode/SOT confirm a scaling range in the G-band spectrum up to 53 mHz also consistent with turbulent motions as well as the identification of two distinct states in terms of the H-line intensity and G-band power as functions of G-band intensity.

Rapid Oscillations in the Solar Atmosphere: Spectra and Physical Effects

High-frequency fluctuations are observed with the Rapid Oscillations in the Solar Atmosphere (ROSA) instrument (Jess et al. 2010, Solar Phys, 261, 363) at the Dunn Solar Telescope. This can produce simultaneous observations in up to six channels, at different heights in the photosphere and chromosphere, at an unprecedentedly high cadence of 0.5 seconds, and at a spatial resolution of 100 km after photometrically correct speckle reconstruction. Here we concentrate on observations at two levels. The first is in the G-band of the CH radical at 4305.5Å, bandpass 9.2Å, with height of formation z <250 km at a cadence of 0.525 sec corresponding to Nyquist frequency 950 mHz. The second is in the Ca II K-line core at 3933.7Å, bandpass 1.0Å, with height of formation z <1300 km, and cadence 4.2 sec giving Nyquist frequency 120 mHz. The data span 53 min, and the maximum field of view is 45 Mm. The data were taken on 28 May 2009 in internetwork and network near disk center. Using both Fourier and Morlet wavelet methods we find evidence in the G-band spectra for intensity fluctuations above noise out to frequencies f >> 100 mHz. The K-line signal is noisier and is seen only for f <50 mHz. With wavelet techniques we find that G-band spectral power with 20 <f <100 mHz is clearly concentrated in the intergranular lanes and especially at the locations of magnetic elements indicated by G-band bright points. This wavelet power is highly intermittent in time. By cross-correlating the data we find that pulses of high-frequency G-band power in the photosphere tend to be followed by increases in K-line emission in the chromosphere with a time lag of about 2 min.