An international patient-centred study of retroperitoneal fibrosis.

BACKGROUND: The impact that rare chronic disorders, such as retroperitoneal fibrosis (RPF), can have on the physical and psychological aspects of a patient's health is poorly understood. Patient-related outcome measures and experiences provide a unique opportunity to understand the impact rare chronic disorders have on a patient's life as well as allowing healthcare providers to compare and improve performance. AIM: To understand the physical and psychosocial impact that RPF has upon peoples' lives. DESIGN: An international online questionnaire was therefore created to gain insights into how patients with RPF, a rare fibro-inflammatory condition, viewed their health and experiences. METHODS: An international online questionnaire comprising 62 questions/free text options, was designed in collaboration with two patient advocates and the multi-disciplinary Renal Association Rare Disease Registry (RaDaR) RPF Group the questionnaire was anonymous and freely accessible on a GOOGLE Form online platform for 6 months. RESULTS: A total of 229 patients from 30 countries across 5 continents responded. Four key issues were identified; (i) pain; (ii) therapy-related side effects; (iii) lack of informed doctors/information about their condition and its management; and (iv) psychological burden. Variations in diagnosis and management are highlighted with 55% undergoing a biopsy to reach a diagnosis of RPF; 75% of patients underwent a further interventional procedure with 60% concurrently treated medically. CONCLUSION: This study will guide further development of clinical and academic multi-disciplinary activity and shows the importance of trying to understand the impact of rare chronic disorders on the physical and psychological aspects of a patient's health.

Rapid detection of Phytophthora ramorum and P. kernoviae by two-minute DNA extraction followed by isothermal amplification and amplicon detection by generic lateral flow device.

ABSTRACT A method for nucleic-acid-based detection of pathogens in plant material has been developed which comprises a simple and rapid method for extracting DNA on the nitrocellulose membranes of lateral-flow devices, loop-mediated isothermal amplification (LAMP) of target DNA using labeled primers, and detection of the generically labeled amplification products by a sandwich immunoassay in a lateral-flow-device format. Each of these steps can be performed without specialist equipment and is suitable for on-site use, and a result can be obtained in just over an hour. A LAMP assay for the detection of plant DNA (cytochrome oxidase gene) can be used in conjunction with pathogen-specific assays to confirm negative results. The use of this method is demonstrated for the detection of Phytophthora ramorum, the causal agent of sudden oak death and dieback/leaf blight in a range of tree, shrub, and herbaceous species, and the recently described pathogen P. kernoviae.

Detection of Botrytis cinerea by loop-mediated isothermal amplification.

AIMS: To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop-mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting. METHODS AND RESULTS: A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross-reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real-time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in <15 min. CONCLUSIONS: The LAMP assay that was developed is suitable for rapid detection of B. cinerea in infected plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: The LAMP method combines the sensitivity and specificity of nucleic acid-based methods with simplified equipment and a reduced reaction time. These features make the method potentially suitable for on-site use, where the results of testing could help to inform decisions regarding the storage and processing of commodities affected by B. cinerea, such as cut flowers, fruit and vegetables.

IgG4-related disease in a multi-ethnic community: clinical characteristics and association with malignancy.

BACKGROUND: Immunoglobulin-G4-related disease (IgG4-RD) is a recently recognized fibro-inflammatory condition that can affect multiple organs. Despite growing interest in this condition, the natural history and management of IgG4-RD remain poorly understood. AIM: To describe the clinical characteristics, treatment and outcomes of IgG4-RD in a multi-ethnic UK cohort, and investigate its possible association with malignancy. DESIGN: Retrospective analysis of case-note and electronic data. METHODS: Cases were identified from sub-specialty cohorts and a systematic search of an NHS trust histopathology database using 'IgG4' or 'inflammatory pseudotumour' as search terms. Electronic records, imaging and histopathology reports were reviewed. RESULTS: In total, 66 identified cases of IgG4-RD showed a similar multi-ethnic spread to the local population of North West London. The median age was 59 years and 71% of patients were male. Presenting symptoms relating to mass effect of a lesion were present in 48% of cases and the mean number of organs involved was 2.4. Total of 10 patients had reported malignancies with 6 of these being haematological. 83% of those treated with steroids had good initial response; however, 50% had relapsing-remitting disease. Rituximab was administered in 11 cases and all achieved an initial serological response. Despite this, seven patients subsequently relapsed after a mean duration of 11 months and four progressed despite treatment. CONCLUSIONS: We report a large UK-based cohort of IgG4-RD that shows no clear ethnic predisposition and a wide range of affected organs. We discuss the use of serum IgG4 concentrations as a disease marker in IgG4-RD, the association with malignant disease and outcomes according to differing treatment regimens.

Detection of clenbuterol in bovine retinal tissue by high-performance liquid chromatography with electrochemical detection.

A method for the detection of the beta-agonist drug clenbuterol in bovine retinal tissue has been developed. The extraction procedure involves sonication and centrifugation, followed by the addition of ethylenediaminetetraacetic acid (EDTA) to the supernatant. The pH of the supernatant is then brought to 12.2, which is then allowed to sit for 2 h. This is followed by a diethylether extraction. The diethylether extract is dried under nitrogen and the residue is dissolved in 1% formic acid. The quantitation of clenbuterol was accomplished by high-performance liquid chromatography with electrochemical detection. The electrochemical detector was an amperometric detection. The detector was set in the pulsed mode. The oxidizing potential of a carbon electrode was 1.3 V vs. a Ag/AgCl reference electrode and was pulsed to a reduction potential of 2.0 V vs. a Ag/AgCl reference electrode. The limit of detection for this method was 5 ng/ml of clenbuterol (S/N = 3). Typical spiked recoveries are 75%.

Malignant fibrous histiocytoma developing in burn scar of the ear.

Malignant fibrous histiocytoma is the most common soft tissue sarcoma. The tumours usually present as a painless enlarging mass deep to the subcutaneous tissue most often in the thigh. Wide excision is the recommended treatment, as the incidence of recurrence is high in patients with marginal excision. Metastases can occur in 50 per cent of patients. This patient represents a rare and previously unreported presentation of malignant fibrous histiocytoma in a burn scar of the ear.

Supermarket tampering: cocaine detected in syringes and in fruit.

Product tampering, as detailed by the Federal Anti-Tampering Act of 1983 (1), is a felony punishable by both fine and imprisonment. The rationale for product tampering ranges from pranks and attention seeking acts to extortion, terrorism, and homicide. One such case submitted for analysis involved four medical syringes found in a supermarket and suspected of being used to tamper with various products. One of the syringes was found piercing a pear while the other three syringes were found with needles exposed in other parts of the supermarket. Microscopic analysis was used to collect residue from the syringe barrels and the pear. A multidiscipline approach involving SLM, PLM, including microchemical analysis, FTIR, and GC/MS analyses, performed on the residual liquid found in the syringe barrels and in the suspect pear, confirmed the presence of cocaine. This multidisciplinary approach is often necessary when there is a possible health risk to the public and rapid response is important. With this approach, it was quickly determined which drugs or poisons were used in this tampering.

Loop-mediated isothermal amplification for rapid detection of the causal agents of cassava brown streak disease.

The causal agents of cassava brown streak disease have recently been identified as Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Primers have been developed for rapid detection of these viruses by reverse transcription loop-mediated isothermal amplification (RT-LAMP). Performance of the RT-LAMP assays compared favourably with published RT-PCR and real-time RT-PCR methods. Furthermore, amplification by RT-LAMP is completed in 40 min and does not require thermal cycling equipment. Modification of the RT-LAMP reactions to use labelled primers allowed rapid detection of amplification products using lateral flow devices containing antibodies specific to the incorporated labels, avoiding the need for fluorescence detection or gel electrophoresis.

A five-minute DNA extraction method for expedited detection of Phytophthora ramorum following prescreening using Phytophthora spp. lateral flow devices.

In a direct comparison with established methods for Phytophthora ramorum detection (isolation followed by morphological identification, or conventional DNA extraction followed by TaqMan real-time PCR) a rapid, simplified detection method in which membranes of lateral flow devices (LFDs) are added directly to TaqMan real-time PCR reactions was used to test 202 plant samples collected by plant health inspectors in the field. P. ramorum prevalence within the 202 samples was approximately 40% according to routine testing by isolation or TaqMan real-time PCR. The diagnostic sensitivity and specificity of the rapid detection method were 96.3% and 91.2%, respectively. This method can be used in conjunction with Phytophthora spp. lateral flow devices to reduce the number of samples requiring testing using more laborious conventional methods. The effect of combining prescreening for Phytophthora spp. with P. ramorum-specific tests is discussed in terms of the positive and negative predictive values of species-specific detection when testing samples collected in different inspection scenarios.

Faster, simpler, more-specific methods for improved molecular detection of Phytophthora ramorum in the field.

Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.

On-site DNA extraction and real-time PCR for detection of Phytophthora ramorum in the field.

Phytophthora ramorum is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of P. ramorum, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for P. ramorum was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a cost-effective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.

Increasing bioanalytical throughput using pcSFC-MS/MS: 10 minutes per 96-well plate.

The utility of packed-column supercritical, subcritical, and enhanced fluidity liquid chromatographies (pcSFC) for high-throughput applications has increased during the past few years. In contrast to traditional reversed-phase liquid chromatography, the addition of a volatile component to the mobile phase, such as CO2, produces a lower mobile-phase viscosity. This allows the use of higher flow rates which can translate into faster analysis times. In addition, the resulting mobile phase is considerably more volatile than the aqueous-based mobile phases that are typically used with LC-MS, allowing the entire effluent to be directed into the MS interface. High-throughput bioanalytical quantitation using pcSFC-MS/MS for pharmacokinetics applications is demonstrated in this report using dextromethorphan as a model compound. Plasma samples were prepared by automated liquid/liquid extraction in the 96-well format prior to pcSFC-MS/MS analysis. Three days of validation data are provided along with study sample data from a patient dosed with commercially available Vicks 44. Using pcSFC and MS/MS, dextromethorphan was quantified in 96-well plates at a rate of approximately 10 min/plate with average intraday accuracy of 9% or better. Daily relative standard deviations (RSDs) were less than 10% for the 2.21 and 14.8 ng/mL quality control (QC) samples, while the RSDs were less than 15% at the 0.554 ng/mL QC level.