Direct and quantitative detection of bacteriophage by “hearing” surface detachment using a quartz crystal microbalance

We show that it is possible to detect specifically adsorbed bacteriophage directly by breaking the interactions between proteins displayed on the phage coat and ligands immobilized on the surface of a quartz crystal microbalance (QCM). This is achieved through increasing the amplitude of oscillation of the QCM surface and sensitively detecting the acoustic emission produced when the bacteriophage detaches from the surface. There is no interference from nonspecifically adsorbed phage. The detection is quantitative over at least 5 orders of magnitude and is sensitive enough to detect as few as 20 phage. The method has potential as a sensitive and low-cost method for virus detection.

Direct and quantitative detection of bacteriophage by "hearing" surface detachment using a quartz crystal microbalance

We show that it is possible to detect specifically adsorbed bacteriophage directly by breaking the interactions between proteins displayed on the phage coat and ligands immobilized on the surface of a quartz crystal microbalance (QCM). This is achieved through increasing the amplitude of oscillation of the QCM surface and sensitively detecting the acoustic emission produced when the bacteriophage detaches from the surface. There is no interference from nonspecifically adsorbed phage. The detection is quantitative over at least 5 orders of magnitude and is sensitive enough to detect as few as 20 phage. The method has potential as a sensitive and low-cost method for virus detection.

Macroscopic fundamental diagram for Brisbane, Australia: empirical findings on network partitioning and incident detection

The Macroscopic Fundamental Diagram (MFD) relates space-mean density and flow, and the existence with dynamic features was confirmed in congested urban network in downtown Yokohama with real data set. Since the MFD represents the area-wide network traffic performances, studies on perimeter control strategies and an area traffic state estimation utilizing the MFD concept has been reported. However, limited works have been reported on real world example from signalised arterial network. This paper fuses data from multiple sources (Bluetooth, Loops and Signals) and presents a framework for the development of the MFD for Brisbane, Australia. Existence of the MFD in Brisbane arterial network is confirmed. Different MFDs (from whole network and several sub regions) are evaluated to discover the spatial partitioning for network performance representation. The findings confirmed the usefulness of appropriate network partitioning for traffic monitoring and incident detections. The discussion addressed future research directions.

Can skin cancer prevention and early detection be improved via mobile phone text messaging? a randomised, attention-control trial.

Objective. To test the impact of a theory-based, SMS (text message)-delivered behavioural intervention (Healthy Text) targeting sun protection or skin self-examination behaviours compared to attention-control. Method. Overall, 546 participants aged 18–42 years were randomised using a computer-generated number list to the skin self-examination (N = 176), sun protection (N = 187), or attention-control (N = 183) text messages group. Each group received 21 text messages about their assigned topic over 12 months (12 weekly messages for three months, then monthly messages for the next nine months). Data was collected via telephone survey at baseline, three-, and 12-months across Queensland from January 2012 to August 2013. Results. One year after baseline, the sun protection (mean change 0.12; P = 0.030) and skin self-examination groups (mean change 0.12; P = 0.035) had significantly greater improvement in their sun protection habits (SPH) index compared to the attention-control group (reference mean change 0.02). The increase in the proportion of participants who reported any skin self-examination from baseline to 12 months was significantly greater in the skin self-examination intervention group (103/163; 63%; P < 0.001) than the sun protection (83/173; 48%), or attention-control (65/165; 36%) groups. There was no significant effect of the intervention for participants who self-reported whole-body skin self-examination, sun tanning behaviour, or sunburn behaviours. Conclusion. The Healthy Text intervention was effective in inducing significant improvements in sun protection and any type of skin self-examination behaviours.

User preferences for text message-delivered skin cancer prevention and early detection

Evidence is needed for the acceptability and user preferences of receiving skin cancer-related text messages. We prepared 27 questions to evaluate attitudes, satisfaction with program characteristics such as timing and spacing, and overall satisfaction with the Healthy Text program in young adults. Within this randomised controlled trial (age 18-42 years), 546 participants were assigned to one of three Healthy Text message groups; sun protection, skin self-examination, or attention-control. Over a 12-month period, 21 behaviour-specific text messages were sent to each group. Participants’ preferences were compared between the two interventions and control group at the 12-month follow-up telephone interview. In all three groups, participants reported the messages were easy to understand (98%), provided good suggestions or ideas (88%), and were encouraging (86%) and informative (85%) with little difference between the groups. The timing of the texts was received positively (92%); however, some suggestions for frequency or time of day the messages were received from 8% of participants. Participants in the two intervention groups found their messages more informative, and triggering behaviour change compared to control. Text messages about skin cancer prevention and early detection are novel and acceptable to induce behaviour change in young adults.

Reproducible and label-free biosensor for the selective extraction and rapid detection of proteins in biological fluids

Erythropoietin (EPO), a glycoprotein hormone of ∼34 kDa, is an important hematopoietic growth factor, mainly produced in the kidney and controls the number of red blood cells circulating in the blood stream. Sensitive and rapid recombinant human EPO (rHuEPO) detection tools that improve on the current laborious EPO detection techniques are in high demand for both clinical and sports industry. A sensitive aptamer-functionalized biosensor (aptasensor) has been developed by controlled growth of gold nanostructures (AuNS) over a gold substrate (pAu/AuNS). The aptasensor selectively binds to rHuEPO and, therefore, was used to extract and detect the drug from horse plasma by surface enhanced Raman spectroscopy (SERS). Due to the nanogap separation between the nanostructures, the high population and distribution of hot spots on the pAu/AuNS substrate surface, strong signal enhancement was acquired. By using wide area illumination (WAI) setting for the Raman detection, a low RSD of 4.92% over 150 SERS measurements was achieved. The significant reproducibility of the new biosensor addresses the serious problem of SERS signal inconsistency that hampers the use of the technique in the field. The WAI setting is compatible with handheld Raman devices. Therefore, the new aptasensor can be used for the selective extraction of rHuEPO from biological fluids and subsequently screened with handheld Raman spectrometer for SERS based in-field protein detection.

Multimodal polymer nanoparticles with combined 19F magnetic resonance and optical detection for tunable, targeted, multimodal imagingin vivo

Understanding the complex nature of diseased tissue in vivo requires development of more advanced nanomedicines, where synthesis of multifunctional polymers combines imaging multimodality with a biocompatible, tunable, and functional nanomaterial carrier. Here we describe the development of polymeric nanoparticles for multimodal imaging of disease states in vivo. The nanoparticle design utilizes the abundant functionality and tunable physicochemical properties of synthetically robust polymeric systems to facilitate targeted imaging of tumors in mice. For the first time, high-resolution 19F/1H magnetic resonance imaging is combined with sensitive and versatile fluorescence imaging in a polymeric material for in vivo detection of tumors. We highlight how control over the chemistry during synthesis allows manipulation of nanoparticle size and function and can lead to very high targeting efficiency to B16 melanoma cells, both in vitro and in vivo. Importantly, the combination of imaging modalities within a polymeric nanoparticle provides information on the tumor mass across various size scales in vivo, from millimeters down to tens of micrometers.

A Bayesian Framework for the Assessment of Vision-based Weed and Fruit Detection and Classification Algorithms

This paper proposes new metrics and a performance-assessment framework for vision-based weed and fruit detection and classification algorithms. In order to compare algorithms, and make a decision on which one to use fora particular application, it is necessary to take into account that the performance obtained in a series of tests is subject to uncertainty. Such characterisation of uncertainty seems not to be captured by the performance metrics currently reported in the literature. Therefore, we pose the problem as a general problem of scientific inference, which arises out of incomplete information, and propose as a metric of performance the(posterior) predictive probabilities that the algorithms will provide a correct outcome for target and background detection. We detail the framework through which these predicted probabilities can be obtained, which is Bayesian in nature. As an illustration example, we apply the framework to the assessment of performance of four algorithms that could potentially be used in the detection of capsicums (peppers).

Characterisation and early detection of an offtype from micropropagated Lady Finger bananas

An offtype has been identified from micropropagated Lady Finger bananas (Musa spp., AAB group, Pome subgroup) that is characterised by its slow growth and poor bunch size. Bunch weights were approximately 25% those of normal Lady Finger plants and all of the fruit produced was unmarketable. This particular offtype is the most commonly encountered from micropropagated Lady Finger plants and, in 2 instances, blocks of 3000 and 1500 plants were entirely comprised of this single offtype. Detection of offtype plants was possible during establishment and growth of plants in the glasshouse by the presence of chlorotic streaks in the leaves. In more severe cases the streaks coalesced into chlorotic patches that developed thin, necrotic areas that eventually produced holes or splits in the leaves. Symptom expression was not ameliorated by the addition of fertiliser and even though symptoms were similar to severe Ca and B deficiency, both normal and offtype plants had similar levels of these elements in the leaves. The offtype plants were also slow growing in the glasshouse and produced significantly (P<0.05) smaller pseudostems and leaves than normal plants. Offtype plants could be readily detected after 4 weeks deflasking using the presence of chlorotic streaks in the leaves as the main selection criterion. Maximum discrimination was possible between weeks 5–7 and at the 6-leaf stage when all of the offtypes could be detected.

Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5' Taq nuclease assay: A new tool for diagnosing infectious coryza

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.

Lantibiotic Nisin and Its Detection Methods

The type A lantibiotic nisin produced by several Lactococcus lactis strains, and one Streptococcus uberis strainis a small antimicrobial peptide that inhibits the growth of a wide range of gram-positive bacteria, such as Bacillus, Clostridium, Listeria and Staphylococcus species. It is nontoxic to humans and used as a food preservative (E234) in more than 50 countries including the EU, the USA, and China. National legislations concerning maximum addition levels of nisin in different foods vary greatly. Therefore, there is a demand for non-laborious and sensitive methods to identify and quantify nisin reliably from different food matrices. The horizontal inhibition assay, based on the inhibitory effect of nisin to Micrococcus luteus is the base for most quantification methods developed so far. However, the sensitivity and accuracy of the agar diffusion method is affected by several parameters. Immunological tests have also been described. Taken into account the sensitivity of immunological methods to interfering substances within sample matrices, and possible cross-reactivities with lantibiotics structurally close to nisin, their usefulness for nisin detection from food samples remains limited. The proteins responsible for nisin biosynthesis, and producer self-immunity are encoded by genes arranged into two inducible operons, nisA/Z/QBTCIPRK and nisFEG, which also contain internal, constitutive promoters PnisI and PnisR. The transmembrane histidine kinase NisK and the response regulator NisR form a two-component signal transduction system, in which NisK autophosphorylates after exposure to extra cellular nisin, and subsequently transfers the phosphate to NisR. The phosphorylated NisR then relays the signal downstream by binding to two regulated promoters in the nisin gene cluster, i.e the nisA/Z/Qand the nisF promoters, thus activating transcription of the structural gene nisA/Z/Q and the downstream genes nisBTCIPRK from the nisA/Z/Q promoter, and the genes nisFEG from the nisF promoter. In this work two novel and highly sensitive nisin bioassays were developed. Both of these quantification methods were based on NisRK mediated, nisin induced Green Fluorescent Protein (GFP) fluorescence. The suitabilities of these assays for quantifica¬tion of nisin from food samples were evaluated in several food matrices. These bioassays had nisin sensitivities in the nanogram or picogram levels. In addition, shelf life of nisin in cooked sausages and retainment of the induction activity of nisin in intestinal chyme (intestinal content) was assessed.

Methicillin-resistant Staphylococcus aureus in Finland: recent changes in the epidemiology, long-term facility aspects, and phenotypic and molecular detection of isolates

Staphylococcus aureus is one of the most important bacteria that cause disease in humans, and methicillin-resistant S. aureus (MRSA) has become the most commonly identified antibiotic-resistant pathogen in many parts of the world. MRSA rates have been stable for many years in the Nordic countries and the Netherlands with a low MRSA prevalence in Europe, but in the recent decades, MRSA rates have increased in those low-prevalence countries as well. MRSA has been established as a major hospital pathogen, but has also been found increasingly in long-term facilities (LTF) and in communities of persons with no connections to the health-care setting. In Finland, the annual number of MRSA isolates reported to the National Infectious Disease Register (NIDR) has constantly increased, especially outside the Helsinki metropolitan area. Molecular typing has revealed numerous outbreak strains of MRSA, some of which have previously been associated with community acquisition. In this work, data on MRSA cases notified to the NIDR and on MRSA strain types identified with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing at the National Reference Laboratory (NRL) in Finland from 1997 to 2004 were analyzed. An increasing trend in MRSA incidence in Finland from 1997 to 2004 was shown. In addition, non-multi-drug resistant (NMDR) MRSA isolates, especially those resistant only to methicillin/oxacillin, showed an emerging trend. The predominant MRSA strains changed over time and place, but two internationally spread epidemic strains of MRSA, FIN-16 and FIN-21, were related to the increase detected most recently. Those strains were also one cause of the strikingly increasing invasive MRSA findings. The rise of MRSA strains with SCCmec types IV or V, possible community-acquired MRSA was also detected. With questionnaires, the diagnostic methods used for MRSA identification in Finnish microbiology laboratories and the number of MRSA screening specimens studied were reviewed. Surveys, which focused on the MRSA situation in long-term facilities in 2001 and on the background information of MRSA-positive persons in 2001-2003, were also carried out. The rates of MRSA and screening practices varied widely across geographic regions. Part of the NMDR MRSA strains could remain undetected in some laboratories because of insufficient diagnostic techniques used. The increasing proportion of elderly population carrying MRSA suggests that MRSA is an emerging problem in Finnish long-term facilities. Among the patients, 50% of the specimens were taken on a clinical basis, 43% on a screening basis after exposure to MRSA, 3% on a screening basis because of hospital contact abroad, and 4% for other reasons. In response to an outbreak of MRSA possessing a new genotype that occurred in a health care ward and in an associated nursing home of a small municipality in Northern Finland in autumn 2003, a point-prevalence survey was performed six months later. In the same study, the molecular epidemiology of MRSA and methicillin-sensitive S. aureus (MSSA) strains were also assessed, the results to the national strain collection compared, and the difficulties of MRSA screening with low-level oxacillin-resistant isolates encountered. The original MRSA outbreak in LTF, which consisted of isolates possessing a nationally new PFGE profile (FIN-22) and internationally rare MLST type (ST-27), was confined. Another previously unrecognized MRSA strain was found with additional screening, possibly indicating that current routine MRSA screening methods may be insufficiently sensitive for strains possessing low-level oxacillin resistance. Most of the MSSA strains found were genotypically related to the epidemic MRSA strains, but only a few of them had received the SCCmec element, and all those strains possessed the new SCCmec type V. In the second largest nursing home in Finland, the colonization of S. aureus and MRSA, and the role of screening sites along with broth enrichment culture on the sensitivity to detect S. aureus were studied. Combining the use of enrichment broth and perineal swabbing, in addition to nostrils and skin lesions swabbing, may be an alternative for throat swabs in the nursing home setting, especially when residents are uncooperative. Finally, in order to evaluate adequate phenotypic and genotypic methods needed for reliable laboratory diagnostics of MRSA, oxacillin disk diffusion and MIC tests to the cefoxitin disk diffusion method at both +35°C and +30°C, both with or without an addition of sodium chloride (NaCl) to the Müller Hinton test medium, and in-house PCR to two commercial molecular methods (the GenoType® MRSA test and the EVIGENETM MRSA Detection test) with different bacterial species in addition to S. aureus were compared. The cefoxitin disk diffusion method was superior to that of oxacillin disk diffusion and to the MIC tests in predicting mecA-mediated resistance in S. aureus when incubating at +35°C with or without the addition of NaCl to the test medium. Both the Geno Type® MRSA and EVIGENETM MRSA Detection tests are usable, accurate, cost-effective, and sufficiently fast methods for rapid MRSA confirmation from a pure culture.

Acoustic emission source location and damage detection in a metallic structure using a graph-theory-based geodesic pproach

A geodesic-based approach using Lamb waves is proposed to locate the acoustic emission (AE) source and damage in an isotropic metallic structure. In the case of the AE (passive) technique, the elastic waves take the shortest path from the source to the sensor array distributed in the structure. The geodesics are computed on the meshed surface of the structure using graph theory based on Dijkstra's algorithm. By propagating the waves in reverse virtually from these sensors along the geodesic path and by locating the first intersection point of these waves, one can get the AE source location. The same approach is extended for detection of damage in a structure. The wave response matrix of the given sensor configuration for the healthy and the damaged structure is obtained experimentally. The healthy and damage response matrix is compared and their difference gives the information about the reflection of waves from the damage. These waves are backpropagated from the sensors and the above method is used to locate the damage by finding the point where intersection of geodesics occurs. In this work, the geodesic approach is shown to be suitable to obtain a practicable source location solution in a more general set-up on any arbitrary surface containing finite discontinuities. Experiments were conducted on aluminum specimens of simple and complex geometry to validate this new method.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.