Rapid bioassay-guided screening of toxic substances in vegetable oils that shorten the life of SHRSP rats

It has been consistently reported that vegetable oils including canola oil have a life shortening effect in Stroke-Prone Spontaneously Hypertensive Rats (SHRSP) and this toxic effect is not due to the fatty acid composition of the oil. Although it is possible that the phytosterol content or type of phytosterol present in vegetable oils may play some role in the life shortening effect observed in SHRSP rats this is still not completely resolved. Furthermore supercritical CO2 fractionation of canola oil with subsequent testing in SHRSP rats identified safe and toxic fractions however, the compounds responsible for life shortening effect were not characterised. The conventional approach to screen toxic substances in oils using rats takes more than six months and involves large number of animals. In this article we describe how rapid bioassay-guided screening could be used to identify toxic substances derived from vegetable oils and/or processed foods fortified with vegetable oils. The technique incorporates sequential fractionation of oils/processed foods and subsequent treatment of human cell lines that can be used in place of animal studies to determine cytotoxicity of the fractions with structural elucidation of compounds of interest determined via HPLC-MS and GC-MS. The rapid bioassay-guided screening proposed would require two weeks to test multiple fractions from oils, compared with six months if animal experiments were used to screen toxic effects. Fractionation of oil before bio-assay enhances the effectiveness of the detection of active compounds as fractionation increases the relative concentration of minor components.

MidiCities : test beds for innovative delivery of urban transformation

Cities globally and nationally are facing a range of daunting challenges to respond to a suite of emerging imperatives including a low carbon future, oil vulnerability, demographic re-composition, and the prospect of unpredictable economic shocks. To pursue a future that is sustainable and resilient requires substantial transformation of existing urban areas and creation of new mechanisms to guide and manage delivery of physical, economic and social changes.

Mid-sized cities provide legible, nimble test beds for exploring cross-disciplinary models and innovative governance and delivery techniques. Australia’s ‘MidiCities’ – home to 4 million urban dwellers frequently overlooked by urban policy or research effort – are emerging as crucibles of innovation and experimentation. Most of these cities retain that essential key ingredient for sustainable urbanism, economic resilience and community identity: a strong, highly legible city centre with a tightly clustered diversity of facilities and functions – the multi-functional activity centre that metropolitan suburban hubs yearn to grow up to become!

These diverse MidiCities are passing a threshold of self-confident sophistication, and are now providing valuable lessons for each other, which could be adopted or adapted by metropolitan cities where scale and complexity can often overwhelm the search for new and appropriate approaches to delivery of rapid change while maintaining clear guidance toward the vision of a ‘preferred’ future. A network of professionals working with Australian and New Zealand MidiCities is coalescing toward a cross-disciplinary platform for exchange of experiences and information, mutual support, improved research and understanding, capacity-building and the refinement of new specialist skills and structures.

Rapid detection of SMARCB1 sequence variation using high resolution melting

Background : Rhabdoid tumors are rare cancers of early childhood arising in the kidney, central nervous system and other organs. The majority are caused by somatic inactivating mutations or deletions affecting the tumor suppressor locus SMARCB1 [OMIM 601607]. Germ-line SMARCB1 inactivation has been reported in association with rhabdoid tumor, epitheloid sarcoma and familial schwannomatosis, underscoring the importance of accurate mutation screening to ascertain recurrence and transmission risks. We describe a rapid and sensitive diagnostic screening method, using high resolution melting (HRM), for detecting sequence variations in SMARCB1. Methods : Amplicons, encompassing the nine coding exons of SMARCB1, flanking splice site sequences and the 5' and 3' UTR, were screened by both HRM and direct DNA sequencing to establish the reliability of HRM as a primary mutation screening tool. Reaction conditions were optimized with commercially available HRM mixes. Results : The false negative rate for detecting sequence variants by HRM in our sample series was zero. Nine amplicons out of a total of 140 (6.4%) showed variant melt profiles that were subsequently shown to be false positive. Overall nine distinct pathogenic SMARCB1 mutations were identified in a total of 19 possible rhabdoid tumors. Two tumors had two distinct mutations and two harbored SMARCB1 deletion. Other mutations were nonsense or frame-shifts. The detection sensitivity of the HRM screening method was influenced by both sequence context and specific nucleotide change and varied from 1: 4 to 1:1000 (variant to wild-type DNA). A novel method involving digital HRM, followed by re-sequencing, was used to confirm mutations in tumor specimens containing associated normal tissue. Conclusions : This is the first report describing SMARCB1 mutation screening using HRM. HRM is a rapid, sensitive and inexpensive screening technology that is likely to be widely adopted in diagnostic laboratories to facilitate whole gene mutation screening.

Emergency department rapid response systems: the case for a standardized approach to deteriorating patients

Objectives: 

Evaluation of the Test-mate ChE (cholinesterase) field kit in acute organophosphorus poisoning

STUDY OBJECTIVE: Measurement of acetylcholinesterase (AChE) is recommended in the management of organophosphorus poisoning, which results in 200,000 deaths worldwide annually. The Test-mate ChE 400 is a portable field kit designed for detecting occupational organophosphorus exposure that measures RBC AChE and plasma cholinesterase (PChE) within 4 minutes. We evaluate Test-mate against a reference laboratory test in patients with acute organophosphorus self-poisoning. METHODS: This was a cross-sectional comparison study of 14 patients with acute organophosphorus poisoning between May 2007 and June 2008. RBC AChE and PChE were measured in 96 and 91 samples, respectively, with the Test-mate ChE field kit and compared with a reference laboratory, using the limits of agreement method (Bland and Altman), κ statistics, and Spearman's correlation coefficients. RESULTS: There was good agreement between the Test-mate ChE and the reference laboratory for RBC AChE. The mean difference (Test-mate-reference) was -0.62 U/g hemoglobin, 95% limits of agreement -10.84 to 9.59 U/g hemoglobin. Good agreement was also observed between the categories of mild, moderate, and severe RBC AChE inhibition (weighted κ 0.85; 95% confidence interval [CI] 0.83 to 0.87). Measurement of PChE also showed good agreement, with a mean difference (Test-mate-reference) of +0.06 U/mL blood, 95% limits of agreement -0.41 to 0.53 U/mL blood. Spearman's correlation coefficients were 0.87 (95% CI 0.81 to 0.91) for RBC AChE and 0.76 (95% CI 0.66 to 0.84) for PChE. Analysis for within-subject correlation of subjects did not change the limits of agreement. CONCLUSION: The Test-mate ChE field kit reliably provides rapid measurement of RBC AChE in acute organophosphorus poisoning.

Foot-and-mouth disease in red deer - experimental infection and test methods performance

Summary: The aim of this study was to evaluate a number of foot-and-mouth disease (FMD) test methods for use in red deer. Ten animals were intranasally inoculated with the FMD virus (FMDV) O UKG 11/2001, monitored for clinical signs, and samples taken regularly (blood, serum, oral swabs, nasal swabs, probang samples and lesion swabs, if present) over a 4-week period. Only one animal, deer 1103, developed clinical signs (lesions under the tongue and at the coronary band of the right hind hoof). It tested positive by 3D and IRES real-time reverse transcription polymerase chain reaction (rRT-PCR) in various swabs, lesion materials and serum. In a non-structural protein (NSP) in-house ELISA (NSP-ELISA-IH), one commercial ELISA (NSP-ELISA-PR) and a commercial antibody NSP pen side test, only deer 1103 showed positive results from day post-inoculation (dpi) 14 onwards. Two other NSP-ELISAs detected anti-NSP serum antibodies with lower sensitivity. It also showed rising antibody levels in the virus neutralization test (VNT), the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR at dpi 9, and in another two commercial SPO-ELISAs at dpi 12 (SPO-ELISA-IV) and dpi 19 (SPO-ELISA-IZ), respectively. Six of the red deer that had been rRT-PCR and antibody negative were re-inoculated intramuscularly with the same O-serotype FMDV at dpi 14. None of these animals became rRT-PCR or NSP-ELISA positive, but all six animals became positive in the VNT, the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR. Two other commercial SPO-ELISAs were less sensitive or failed to detect animals as positive. The rRT-PCRs and the four most sensitive commercial ELISAs that had been used for the experimentally inoculated deer were further evaluated for diagnostic specificity (DSP) using 950 serum samples and 200 nasal swabs from non-infected animals. DSPs were 100% for the rRT-PCRs and between 99.8 and 100% for the ELISAs.

Application of the Ceditest® FMDV type O and FMDV-NS enzyme-linked immunosorbent assays for detection of antibodies against foot-and-mouth disease virus in selected livestock and wildlife species in Uganda

Diagnosis and control of Foot-and-mouth disease virus (FMDV) requires rapid and sensitive diagnostic tests. Two antibody enzyme-linked immunosorbent assay (ELISA) kits, Ceditest® FMDV-NS for the detection of antibodies against the nonstructural proteins of all FMDV serotypes and Ceditest® FMDV type O for the detection of antibodies against serotype O, were evaluated under African endemic conditions where the presence of multiple serotypes and the use of nonpurified vaccines complicate serological diagnosis. Serum samples from 218 African buffalo, 758 cattle, 304 goats, and 88 sheep were tested using both kits, and selected samples were tested not only in serotype-specific ELISAs for antibodies against primarily FMDV serotype O, but also against other serotypes. The FMDV-NS assay detected far more positive samples (93%) than the FMDV type O assay (30%) in buffalo (P < 0.05), with predominant antibodies against the South African Territories (SAT) serotypes, while the seroprevalence was generally comparable in cattle with antibodies against serotype O elicited by infection and/or vaccination. However, some districts had higher seroprevalence using the FMDV type O assay indicating vaccination without infection, while 1 cattle herd with antibodies against the SAT serotypes had far more positive samples (85%) using the FMDV-NS versus the FMDV type O (10%), consistent with the latter test's lower sensitivity for antibodies against SAT serotypes. Based on the current investigation, the FMDV type O ELISA may be limited by the presence of SAT serotypes. The FMD NS assay worked well as a screening test for antibodies against all FMDV serotypes present in Uganda; however, as long as nonpurified vaccines are applied in the region, this test cannot be used to differentiate between vaccinated and infected animals.

Rapid effects of essential fatty acid deficiency on growth and development parameters and transcription of key fatty acid metabolism genes in juvenile barramundi (Lates calcarifer)

Barramundi (Lates calcarifer), a catadromous teleost of significant and growing commercial importance, are reported to have limited fatty acid bioconversion capability and therefore require preformed long-chain PUFA (LC-PUFA) as dietary essential fatty acid (EFA). In this study, the response of juvenile barramundi (47·0 g/fish initial weight) fed isolipidic and isoenergetic diets with 8·2 % added oil was tested. The experimental test diets were either devoid of fish oil (FO), and thus with no n-3 LC-PUFA (FO FREE diet), or with a low inclusion of FO (FO LOW diet). These were compared against a control diet containing only FO (FO CTRL diet) as the added lipid source, over an 8-week period. Interim samples and measurements were taken fortnightly during the trial in order to define the aetiology of the onset and progression of EFA deficiency. After 2 weeks, the fish fed the FO FREE and FO LOW diets had significantly lower live-weights, and after 8 weeks significant differences were detected for all performance parameters. The fish fed the FO FREE diet also had a significantly higher incidence of external abnormalities. The transcription of several genes involved in fatty acid metabolism was affected after 2 weeks of feeding, showing a rapid nutritional regulation. This experiment documents the aetiology of the onset and the progression of EFA deficiency in juvenile barramundi and demonstrates that such deficiencies can be detected within 2 weeks in juvenile fish.

Deploying aptameric sensing technology for rapid pandemic monitoring

The genome of virulent strains may possess the ability to mutate by means of antigenic shift and/or antigenic drift as well as being resistant to antibiotics with time. The outbreak and spread of these virulent diseases including avian influenza (H1N1), severe acute respiratory syndrome (SARS-Corona virus), cholera (Vibrio cholera), tuberculosis (Mycobacterium tuberculosis), Ebola hemorrhagic fever (Ebola Virus) and AIDS (HIV-1) necessitate urgent attention to develop diagnostic protocols and assays for rapid detection and screening. Rapid and accurate detection of first cases with certainty will contribute significantly in preventing disease transmission and escalation to pandemic levels. As a result, there is a need to develop technologies that can meet the heavy demand of an all-embedded, inexpensive, specific and fast biosensing for the detection and screening of pathogens in active or latent forms to offer quick diagnosis and early treatments in order to avoid disease aggravation and unnecessary late treatment costs. Nucleic acid aptamers are short, single-stranded RNA or DNA sequences that can selectively bind to specific cellular and biomolecular targets. Aptamers, as new-age bioaffinity probes, have the necessary biophysical characteristics for improved pathogen detection. This article seeks to review global pandemic situations in relation to advances in pathogen detection systems. It particularly discusses aptameric biosensing and establishes application opportunities for effective pandemic monitoring. Insights into the application of continuous polymeric supports as the synthetic base for aptamer coupling to provide the needed convective mass transport for rapid screening is also presented.

The diagnostic validity and reliability of an internet-based clinical assessment program for mental disorders

BACKGROUND: Internet-based assessment has the potential to assist with the diagnosis of mental health disorders and overcome the barriers associated with traditional services (eg, cost, stigma, distance). Further to existing online screening programs available, there is an opportunity to deliver more comprehensive and accurate diagnostic tools to supplement the assessment and treatment of mental health disorders. OBJECTIVE: The aim was to evaluate the diagnostic criterion validity and test-retest reliability of the electronic Psychological Assessment System (e-PASS), an online, self-report, multidisorder, clinical assessment and referral system. METHODS: Participants were 616 adults residing in Australia, recruited online, and representing prospective e-PASS users. Following e-PASS completion, 158 participants underwent a telephone-administered structured clinical interview and 39 participants repeated the e-PASS within 25 days of initial completion. RESULTS: With structured clinical interview results serving as the gold standard, diagnostic agreement with the e-PASS varied considerably from fair (eg, generalized anxiety disorder: κ=.37) to strong (eg, panic disorder: κ=.62). Although the e-PASS' sensitivity also varied (0.43-0.86) the specificity was generally high (0.68-1.00). The e-PASS sensitivity generally improved when reducing the e-PASS threshold to a subclinical result. Test-retest reliability ranged from moderate (eg, specific phobia: κ=.54) to substantial (eg, bulimia nervosa: κ=.87). CONCLUSIONS: The e-PASS produces reliable diagnostic results and performs generally well in excluding mental disorders, although at the expense of sensitivity. For screening purposes, the e-PASS subclinical result generally appears better than a clinical result as a diagnostic indicator. Further development and evaluation is needed to support the use of online diagnostic assessment programs for mental disorders. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry ACTRN121611000704998; http://www.anzctr.org.au/trial_view.aspx?ID=336143 (Archived by WebCite at http://www.webcitation.org/618r3wvOG).

Rapid directional change degrades GPS distance measurement validity during intermittent intensity running

Use of the Global Positioning System (GPS) for quantifying athletic performance is common in many team sports. The effect of running velocity on measurement validity is well established, but the influence of rapid directional change is not well understood in team sport applications. This effect was systematically evaluated using multidirectional and curvilinear adaptations of a validated soccer simulation protocol that maintained identical velocity profiles. Team sport athletes completed 90 min trials of the Loughborough Intermittent Shuttle-running Test movement pattern on curvilinear, and multidirectional shuttle running tracks while wearing a 5 Hz (with interpolated 15 Hz output) GPS device. Reference total distance (13 200 m) was systematically over- and underestimated during curvilinear (2.61±0.80%) and shuttle (-3.17±2.46%) trials, respectively. Within-epoch measurement uncertainty dispersion was widest during the shuttle trial, particularly during the jog and run phases. Relative measurement reliability was excellent during both trials (Curvilinear r = 1.00, slope = 1.03, ICC = 1.00; Shuttle r = 0.99, slope = 0.97, ICC = 0.99). Absolute measurement reliability was superior during the curvilinear trial (Curvilinear SEM = 0 m, CV = 2.16%, LOA ± 223 m; Shuttle SEM = 119 m, CV = 2.44%, LOA ± 453 m). Rapid directional change degrades the accuracy and absolute reliability of GPS distance measurement, and caution is recommended when using GPS to quantify rapid multidirectional movement patterns.

The impact of a line probe assay based diagnostic algorithm on time to treatment initiation and treatment outcomes for multidrug resistant TB patients in Arkhangelsk region, Russia

BACKGROUND: In the Arkhangelsk region of Northern Russia, multidrug-resistant (MDR) tuberculosis (TB) rates in new cases are amongst the highest in the world. In 2014, MDR-TB rates reached 31.7% among new cases and 56.9% among retreatment cases. The development of new diagnostic tools allows for faster detection of both TB and MDR-TB and should lead to reduced transmission by earlier initiation of anti-TB therapy. STUDY AIM: The PROVE-IT (Policy Relevant Outcomes from Validating Evidence on Impact) Russia study aimed to assess the impact of the implementation of line probe assay (LPA) as part of an LPA-based diagnostic algorithm for patients with presumptive MDR-TB focusing on time to treatment initiation with time from first-care seeking visit to the initiation of MDR-TB treatment rather than diagnostic accuracy as the primary outcome, and to assess treatment outcomes. We hypothesized that the implementation of LPA would result in faster time to treatment initiation and better treatment outcomes.

METHODS: A culture-based diagnostic algorithm used prior to LPA implementation was compared to an LPA-based algorithm that replaced BacTAlert and Löwenstein Jensen (LJ) for drug sensitivity testing. A total of 295 MDR-TB patients were included in the study, 163 diagnosed with the culture-based algorithm, 132 with the LPA-based algorithm.

RESULTS: Among smear positive patients, the implementation of the LPA-based algorithm was associated with a median decrease in time to MDR-TB treatment initiation of 50 and 66 days compared to the culture-based algorithm (BacTAlert and LJ respectively, p<0.001). In smear negative patients, the LPA-based algorithm was associated with a median decrease in time to MDR-TB treatment initiation of 78 days when compared to the culture-based algorithm (LJ, p<0.001). However, several weeks were still needed for treatment initiation in LPA-based algorithm, 24 days in smear positive, and 62 days in smear negative patients. Overall treatment outcomes were better in LPA-based algorithm compared to culture-based algorithm (p = 0.003). Treatment success rates at 20 months of treatment were higher in patients diagnosed with the LPA-based algorithm (65.2%) as compared to those diagnosed with the culture-based algorithm (44.8%). Mortality was also lower in the LPA-based algorithm group (7.6%) compared to the culture-based algorithm group (15.9%). There was no statistically significant difference in smear and culture conversion rates between the two algorithms.

CONCLUSION: The results of the study suggest that the introduction of LPA leads to faster time to MDR diagnosis and earlier treatment initiation as well as better treatment outcomes for patients with MDR-TB. These findings also highlight the need for further improvements within the health system to reduce both patient and diagnostic delays to truly optimize the impact of new, rapid diagnostics.