Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking log of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value = 1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region. (C) 2012 Elsevier B.V. All rights reserved.

Applicability of the van der Pauw-Hall measurement technique to implanted samples

The application of the van der Pauw-Hall measurement technique to implanted samples in which the mobility varies with depth has still not been fully justified. A proof that the technique is in fact applicable in this situation is given. Journal of Applied Physics is copyrighted by The American Institute of Physics.

Monitoring exposure to polycyclic aromatic hydrocarbons in an Australian population using pooled urine samples

Integrated exposure to polycyclic aromatic hydrocarbons (PAHs) can be assessed through monitoring of urinary mono-hydroxylated PAHs (OH-PAHs). The aim of this study was to provide the first assessment of exposure to PAHs in a large sample of the population in Queensland, Australia including exposure to infant (0-4. years). De-identified urine specimens, obtained from a pathology laboratory, were stratified by age and sex, and pooled (n. =. 24 pools of 100) and OH-PAHs were measured by gas chromatography-isotope dilution-tandem mass spectrometry. Geometric mean (GM) concentrations ranged from 30. ng/L (4-hydroxyphenanthrene) to 9221. ng/L (1-naphthol). GM of 1-hydroxypyrene, the most commonly used PAH exposure biomarker, was 142. ng/L. The concentrations of OH-PAHs found in this study are consistent with those in developed countries and lower than those in developing countries. We observed no association between sex and OH-PAH concentrations. However, we observed lower urinary concentrations of all OH-PAHs in samples from infants (0-4. years), children (5-14. years) and the elderly (>. 60. year old) compared with samples from other age groups (15-29, 30-44 and 45-59. years) which may be attributed to age-dependent behaviour-specific exposure sources.

Use of pooled samples to assess human exposure to parabens, benzophenone-3 and triclosan in Queensland, Australia

Parabens, benzophenone-3 and triclosan are common ingredients used as preservatives, ultraviolet radiation filters and antimicrobial agents, respectively. Human exposure occurs through consumption of processed food and use of cosmetics and consumer products. The aim of this study was to provide a preliminary characterisation of exposure to selected personal care product chemicals in the general Australian population. De-identified urine specimens stratified by age and sex were obtained from a community-based pathology laboratory and pooled (n= 24 pools of 100). Concentrations of free and total (sum of free plus conjugated) species of methyl, ethyl, propyl and butyl paraben, benzophenone-3 and triclosan were quantified using isotope dilution tandem mass spectrometry; with geometric means 232, 33.5, 60.6, 4.32, 61.5 and 87.7. ng/mL, respectively. Age was inversely associated with paraben concentration, and females had concentrations approximately two times higher than males. Total paraben and benzophenone-3 concentrations are significantly higher than reported worldwide, and the average triclosan concentration was more than one order of magnitude higher than in many other populations. This study provides the first data on exposure of the general Australian population to a range of common personal care product chemical ingredients, which appears to be prevalent and warrants further investigation.

Persistent organic pollutants in matched breast milk and infant faeces samples

Assessing blood concentration of persistent organic pollutants (POPs) in infants is difficult due to the ethical and practical difficulties in obtaining sufficient quantities of blood. To determine whether measuring POPs in faeces might reflect blood concentration during infancy, we measured the concentrations of a range of POPs (i.e. polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and organochlorine pesticides (OCPs)) in a pilot study using matched breast milk and infant faecal samples obtained from ten mother-child pairs. All infants were breast fed, with 8 of them also receiving solid food at the time of faecal sampling. In this small dataset faecal concentrations (range 0.01-41ngg-1 lipid) are strongly associated with milk concentrations (range 0.02-230ngg-1 lipid). Associations with other factors generally could not be detected in this dataset, with the exception of a small effect of age or growth. Different sources (external or internal) of exposure appeared to directly influence faecal concentrations of different chemicals based on different inter-individual variability in the faeces-to-milk concentration ratio Rfm. Overall, the matrix of faeces as an external measure of internal exposure in infants looks promising for some chemicals and is worth assessing further in larger datasets.

Development of analytical methods for the separation of plutonium, americium, curium and neptunium from environmental samples

In this work, separation methods have been developed for the analysis of anthropogenic transuranium elements plutonium, americium, curium and neptunium from environmental samples contaminated by global nuclear weapons testing and the Chernobyl accident. The analytical methods utilized in this study are based on extraction chromatography. Highly varying atmospheric plutonium isotope concentrations and activity ratios were found at both Kurchatov (Kazakhstan), near the former Semipalatinsk test site, and Sodankylä (Finland). The origin of plutonium is almost impossible to identify at Kurchatov, since hundreds of nuclear tests were performed at the Semipalatinsk test site. In Sodankylä, plutonium in the surface air originated from nuclear weapons testing, conducted mostly by USSR and USA before the sampling year 1963. The variation in americium, curium and neptunium concentrations was great as well in peat samples collected in southern and central Finland in 1986 immediately after the Chernobyl accident. The main source of transuranium contamination in peats was from global nuclear test fallout, although there are wide regional differences in the fraction of Chernobyl-originated activity (of the total activity) for americium, curium and neptunium.

Critical review on the stability of illicit drugs in sewers and wastewater samples

Wastewater-based epidemiology (WBE) applies advanced analytical methods to quantify drug residues in wastewater with the aim to estimate illicit drug use at the population level. Transformation processes during transport in sewers (chemical and biological reactors) and storage of wastewater samples before analysis are expected to change concentrations of different drugs to varying degrees. Ignoring transformation for drugs with low to medium stability will lead to an unknown degree of systematic under- or overestimation of drug use, which should be avoided. This review aims to summarize the current knowledge related to the stability of commonly investigated drugs and, furthermore, suggest a more effective approach to future experiments. From over 100 WBE studies, around 50 mentioned the importance of stability and 24 included tests in wastewater. Most focused on in-sample stability (i.e., sample preparation, preservation and storage) and some extrapolated to in-sewer stability (i.e., during transport in real sewers). While consistent results were reported for rather stable compounds (e.g., MDMA and methamphetamine), a varying range of stability under different or similar conditions was observed for other compounds (e.g., cocaine, amphetamine and morphine). Wastewater composition can vary considerably over time, and different conditions prevail in different sewer systems. In summary, this indicates that more systematic studies are needed to: i) cover the range of possible conditions in sewers and ii) compare results more objectively. To facilitate the latter, we propose a set of parameters that should be reported for in-sewer stability experiments. Finally, a best practice of sample collection, preservation, and preparation before analysis is suggested in order to minimize transformation during these steps.

A novel fully validated LC–MS/MS method for quantification of pyridoxal-5′-phosphate concentrations in samples of human whole blood

Quantification of pyridoxal-5´-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC-MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20 µL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC-MS/MS system. Calibration curves were linear and recovery was > 92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17h or at -80 °C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC-MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.

Prevalence of multidrug-resistant Staphylococcus aureus in diabetics clinical samples

Antibiotic resistance in 40 Staphylococcus aureus clinical isolates from 110 diabetic patients (36%) was evaluated. Of these, 32 (80%) of the isolates showed multidrug-resistance to more than eight antibiotics and 35% isolates were found to be methicillin resistant S. aureus (MRSA). All 40 S. aureus strains (100%) screened from diabetic clinical specimens were resistant to penicillin, 63% to ampicillin, 55% to streptomycin, 50% to tetracycline and 50% to gentamicin. Where as low resistance rate was observed to ciprofloxacin (20%) and rifampicin (8%). In contrast, all (100%) S. aureus strains recorded susceptibility to teicoplanin, which was followed by vancomycin (95%). Genotypical examination revealed that 80% of the aminoglycoside resistant S. aureus (ARSA) have aminoglycoside modifying enzyme (AME) coding genes; however, 20% of ARSA which showed non-AME mediated (adaptive) aminoglycoside resistance lacked these genes in their genome. In contrast all MRSA isolates possessed mecA, femA genetic determinants in their genome.

Learning to write: Analysing writing samples as part of considering how children become writers

This chapter draws on a large data set of children's work samples collected as part of a five-year school reform project in a community of high poverty. One component of the data set from this project is a corpus of more than 2000 writing samples collected from students across eight grade levels (Prep to year 7) annually, across four years of the project (2009-2013). This paper utilises a selection of these texts to consider insights available to teachers and schools through a simple process of collecting and assessing writing samples produced by children over time. The focus is on what samples of writing might enable us to know and understand about learning and teaching this important dimension of literacy in current classrooms.

Metabolomic and proteomic analysis of breast cancer patient samples suggests that glutamate and 12-HETE in combination with CA15-3 may be useful biomarkers reflecting tumour burden

Evaluation of protein and metabolite expression patterns in blood using mass spectrometry and high-throughput antibody-based screening platforms has potential for the discovery of new biomarkers for managing breast cancer patient treatment. Previously identified blood-based breast cancer biomarkers, including cancer antigen 15.3 (CA15-3) are useful in combination with imaging (computed tomography scans, magnetic resonance imaging, X-rays) and physical examination for monitoring tumour burden in advanced breast cancer patients. However, these biomarkers suffer from insufficient levels of accuracy and with new therapies available for the treatment of breast cancer, there is an urgent need for reliable, non-invasive biomarkers that measure tumour burden with high sensitivity and specificity so as to provide early warning of the need to switch to an alternative treatment. The aim of this study was to identify a biomarker signature of tumour burden using cancer and non-cancer (healthy controls/non-malignant breast disease) patient samples. Results demonstrate that combinations of three candidate biomarkers from Glutamate, 12-Hydroxyeicosatetraenoic acid, Beta-hydroxybutyrate, Factor V and Matrix metalloproteinase-1 with CA15-3, an established biomarker for breast cancer, were found to mirror tumour burden, with AUC values ranging from 0.71 to 0.98 when comparing non-malignant breast disease to the different stages of breast cancer. Further validation of these biomarker panels could potentially facilitate the management of breast cancer patients, especially to assess changes in tumour burden in combination with imaging and physical examination.