VapC proteins from Mycobacterium tuberculosis share ribonuclease sequence specificity but differ in regulation and toxicity.

The chromosome of Mycobacterium tuberculosis (Mtb) contains a large number of Type II toxin-antitoxin (TA) systems. The majority of these belong to the VapBC TA family, characterised by the VapC protein consisting of a PIN domain with four conserved acidic residues, and proposed ribonuclease activity. Characterisation of five VapC (VapC1, 19, 27, 29 and 39) proteins from various regions of the Mtb chromosome using a combination of pentaprobe RNA sequences and mass spectrometry revealed a shared ribonuclease sequence-specificity with a preference for UAGG sequences. The TA complex VapBC29 is auto-regulatory and interacts with inverted repeat sequences in the vapBC29 promoter, whereas complexes VapBC1 and VapBC27 display no auto-regulatory properties. The difference in regulation could be due to the different properties of the VapB proteins, all of which belong to different VapB protein families. Regulation of the vapBC29 operon is specific, no cross-talk among Type II TA systems was observed. VapC29 is bacteriostatic when expressed in Mycobacterium smegmatis, whereas VapC1 and VapC27 displayed no toxicity upon expression in M. smegmatis. The shared sequence specificity of the five VapC proteins characterised is intriguing, we propose that the differences observed in regulation and toxicity is the key to understanding the role of these TA systems in the growth and persistence of Mtb.

Evaluation of the first pharmacist-administered vaccinations in Western Australia: a mixed-methods study.

OBJECTIVES: This study evaluated the uptake of Western Australian (WA) pharmacist vaccination services, the profiles of consumers being vaccinated and the facilitators and challenges experienced by pharmacy staff in the preparation, implementation and delivery of services. DESIGN: Mixed-methods methodology with both quantitative and qualitative data through surveys, pharmacy computer records and immuniser pharmacist interviews. SETTING: Community pharmacies in WA that provided pharmacist vaccination services between March and October 2015. PARTICIPANTS: Immuniser pharmacists from 86 pharmacies completed baseline surveys and 78 completed exit surveys; computer records from 57 pharmacies; 25 immuniser pharmacists were interviewed. MAIN OUTCOME MEASURES: Pharmacy and immuniser pharmacist profiles; pharmacist vaccination services provided and consumer profiles who accessed services. RESULTS: 15 621 influenza vaccinations were administered by immuniser pharmacists at 76 WA community pharmacies between March and October 2015. There were no major adverse events, and <1% of consumers experienced minor events which were appropriately managed. Between 12% and 17% of consumers were eligible to receive free influenza vaccinations under the National Immunisation Program but chose to have it at a pharmacy. A high percentage of vaccinations was delivered in rural and regional areas indicating that provision of pharmacist vaccination services facilitated access for rural and remote consumers. Immuniser pharmacists reported feeling confident in providing vaccination services and were of the opinion that services should be expanded to other vaccinations. Pharmacists also reported significant professional satisfaction in providing the service. All participating pharmacies intended to continue providing influenza vaccinations in 2016. CONCLUSIONS: This initial evaluation of WA pharmacist vaccination services showed that vaccine delivery was safe. Convenience and accessibility were important aspects in usage of services. There is scope to expand pharmacist vaccination services to other vaccines and younger children; however, government funding to pharmacists needs to be considered.

Choppers to optimise the repetition rate multiplication technique on a direct geometry neutron chopper spectrometer.

In recent years the use of repetition rate multiplication (RRM) on direct geometry neutron spectrometers has been established and is the common mode of operation on a growing number of instruments. However, the chopper configurations are not ideally optimised for RRM with a resultant 100 fold flux difference across a broad wavelength band. This paper presents chopper configurations that will produce a relative constant (RC) energy resolution and a relative variable (RV) energy resolution for optimised use of RRM. The RC configuration provides an almost uniform ΔE/E for all incident wavelengths and enables an efficient use of time as the entire dynamic range is probed with equivalent statistics, ideal for single shot measurements of transient phenomena. The RV energy configuration provides an almost uniform opening time at the sample for all incident wavelengths with three orders of magnitude in time resolution probed for a single European Spallation Source (ESS) period, which is ideal to probe complex relaxational behaviour. These two chopper configurations have been simulated for the Versatile Optimal Resolution direct geometry spectrometer, VOR, that will be built at ESS.

Enzyme-linked immunosorbent assay detection of melamine in infant formula and wheat food products.

The adulteration of food products with melamine to inflate the nitrogen content necessitates the establishment of analytical methods that can distinguish between proteinaceous ingredients and such adulterants. The specificity and ability to detect melamine by two commercial enzyme-linked immunosorbent assay (ELISA) kits were evaluated along with three protocols for sample preparation. Both ELISAs displayed cross-reactivity with ammeline, but neither was able to detect ammelide or cyanuric acid, indicating either a requirement for the 4,6-diamino-1,3,5-triazine structure or inability to bind 1,3,5-triazine-4,6-diones. The limits of detection for melamine in powder infant formula ranged from 0.2 to 3 microg/g depending on the ELISA kit and the method used to prepare the sample. The limits of detection for melamine in liquid infant formula and wheat products were <1 microg/ml and <2.5 microg/g, respectively. The ELISA kits provide an effective alternative for the analysis of samples suspected of containing melamine without relying on extensive sample preparation or expensive instrumentation.

Performance Tested Method multiple laboratory validation study of ELISA-based assays for the detection of peanuts in food.

Performance Tested Method multiple laboratory validations for the detection of peanut protein in 4 different food matrixes were conducted under the auspices of the AOAC Research Institute. In this blind study, 3 commercially available ELISA test kits were validated: Neogen Veratox for Peanut, R-Biopharm RIDASCREEN FAST Peanut, and Tepnel BioKits for Peanut Assay. The food matrixes used were breakfast cereal, cookies, ice cream, and milk chocolate spiked at 0 and 5 ppm peanut. Analyses of the samples were conducted by laboratories representing industry and international and U.S governmental agencies. All 3 commercial test kits successfully identified spiked and peanut-free samples. The validation study required 60 analyses on test samples at the target level 5 microg peanut/g food and 60 analyses at a peanut-free level, which was designed to ensure that the lower 95% confidence limit for the sensitivity and specificity would not be <90%. The probability that a test sample contains an allergen given a prevalence rate of 5% and a positive test result using a single test kit analysis with 95% sensitivity and 95% specificity, which was demonstrated for these test kits, would be 50%. When 2 test kits are run simultaneously on all samples, the probability becomes 95%. It is therefore recommended that all field samples be analyzed with at least 2 of the validated kits.

Variation of analytical results for peanuts in energy bars and milk chocolate.

Peanuts contain proteins that can cause severe allergic reactions in some sensitized individuals. Studies were conducted to determine the percentage of recovery by an enzyme-linked immunosorbent assay (ELISA) method in the analysis for peanuts in energy bars and milk chocolate and to determine the sampling, subsampling, and analytical variances associated with testing energy bars and milk chocolate for peanuts. Food products containing chocolate were selected because their composition makes sample preparation for subsampling difficult. Peanut-contaminated energy bars, noncontaminated energy bars, incurred milk chocolate containing known levels of peanuts, and peanut-free milk chocolate were used. A commercially available ELISA kit was used for analysis. The sampling, sample preparation, and analytical variances associated with each step of the test procedure to measure peanut protein were determined for energy bars. The sample preparation and analytical variances were determined for milk chocolate. Variances were found to be functions of peanut concentration. Sampling and subsampling variability associated with energy bars accounted for 96.6% of the total testing variability. Subsampling variability associated with powdered milk chocolate accounted for >60% of the total testing variability. The variability among peanut test results can be reduced by increasing sample size, subsample size, and number of analyses. For energy bars the effect of increasing sample size from 1 to 4 bars, subsample size from 5 to 20 g, and number of aliquots quantified from 1 to 2 on reducing the sampling, sample preparation, and analytical variance was demonstrated. For powdered milk chocolate, the effects of increasing subsample size from 5 to 20 g and number of aliquots quantified from 1 to 2 on reducing sample preparation and analytical variances were demonstrated. This study serves as a template for application to other foods, and for extrapolation to different sizes of samples and subsamples as well as numbers of analyses.

Preparation of peanut butter suspension for determination of peanuts using enzyme-linked immunoassay kits.

Peanuts are one of the 8 most common allergenic foods and a large proportion of peanut-allergic individuals have severe reactions, some to minimal exposure. Specific protein constituents in the peanuts are the cause of the allergic reactions in sensitized individuals who ingest the peanuts. To avoid accidental ingestion of peanut-contaminated food, methods of analysis for the determination of the allergenic proteins in foods are important tools. Such methods could help identify foods inadvertently contaminated with peanuts, thereby reducing the incidence of allergic reactions to peanuts. Commercial immunoassay kits are available but need study for method performance, which requires reference materials for within- and between-laboratory validations. In this study, National Institute of Standards and Technology Standard Reference Material 2387 peanut butter was used. A polytron homogenizer was used to prepare a homogenous aqueous Peanut Butter suspension for the evaluation of method performance of some commercially available immunoassay kits such as Veratox for Peanut Allergen Test (Neogen Corp.), Ridascreen Peanut (R-Biopharm GmbH), and Bio-Kit Peanut Protein Assay Kit (Tepnel). Each gram of the aqueous peanut butter suspension contained 20 mg carboxymethylcellulose sodium salt, 643 microg peanut, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. The suspension was homogenous, stable, reproducible, and applicable for adding to ice cream, cookies, breakfast cereals, and chocolate for recovery studies at spike levels ranging from 12 to 90 microg/g.

Community acquisition of gentamicin-sensitive methicillin-resistant Staphylococcus aureus in southeast Queensland, Australia.

Community-acquired methicillin-resistant Staphylococcus aureus (MRSA) susceptible to gentamicin has been reported in a number of countries in the 1990s. To study the acquisition of gentamicin-sensitive MRSA (GS-MRSA) in southeast Queensland and the relatedness of GS-MRSA to other strains of MRSA, 35 cases of infection due to GS-MRSA from October 1997 through September 1998 were examined retrospectively to determine the mode of acquisition and risk factors for MRSA acquisition. Thirty-one isolates from the cases were examined using a variety of methods (antibiotyping, phage typing, pulsed-field gel electrophoresis [PFGE] fingerprinting, and coagulase typing by restriction analysis of PCR products) and were compared with strains of local hospital-acquired gentamicin-resistant MRSA (GR-MRSA) and of Western Australian MRSA (WA-MRSA). Only 6 of 23 cases of community-acquired GS-MRSA had risk factors for MRSA acquisition. Twenty of 21 isolates from cases of community-acquired infection were found to be related by PFGE and coagulase typing and had similar phage typing patterns. Hospital- and nursing home-acquired GS-MRSA strains were genetically and phenotypically diverse. Community-acquired GS-MRSA strains were not related to nosocomial GR-MRSA or WA-MRSA, but phage typing results suggest that they are related to GS-MRSA previously reported in New Zealand.

An epidemiological survey of methicillin-resistant Staphylococcus aureus in a tertiary referral hospital.

Over a 30-month period from July 1995 to December 1997, new detections of methicillin-resistant Staphylococcus aureus (MRSA) were prospectively studied in a tertiary referral hospital. The aims of the study were to determine the incidence of colonization of patients admitted to each of the hospital's 39 clinical units and ascertain where each patient had become colonized. Epidemiological information (time to detection, ward movement, admission to other hospitals, data on MRSA isolations in hospital wards) and phage typing were used by the hospital's infection control unit to make this determination. Routine containment procedures included cohorting, flagging and triclosan body washes. Surveillance cultures were collected infrequently. Patients known to be colonized with MRSA were excluded from orthopaedic and haematology wards. During the study period, 995 patients were found to be newly colonized. The incidence of colonization varied from nil to 72 per 1000 admissions, being highest in the main intensive care unit and in services which frequently used that unit. The incidence of colonization in elective orthopaedic surgery (< 1 per 1000) and haematology (3 per 1000) was very low. Determining the place where patients acquired MRSA was made difficult by the high frequency of endemic phage types and frequent patient transfer between wards. Epidemiological data suggested that the main intensive care unit and surgical wards nursing patients with colorectal, urological and vascular diseases were the places where most patients became colonized. MRSA was never acquired by patients nursed in wards which practised an exclusion policy towards patients known to be colonized with MRSA. Our data suggest that in tertiary referral hospitals, where MRSA is not only endemic but frequently imported from other hospitals, it is possible to establish areas where MRSA is never acquired.

A prospective survey of current methicillin-resistant Staphylococcus aureus control measures.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is now endemic in tertiary referral hospitals among the developed world. By prospective survey, the effect of two measures aimed to reduce the spread of MRSA was determined. First, a surgical ward with persistently high levels of MRSA detection was cleaned and renovated. Second, the medical records of all MRSA-colonized patients were electronically flagged, facilitating immediate application of control measures on readmission. METHODS: Data were collected for 995 newly colonized patients admitted between 1 July 1995 and 31 December 1997. Methicillin-resistant Staphylococcus aureus detection was determined before and after implementation of the interventions, along with the likely place of MRSA acquisition and the monthly incidence of MRSA detection for all inpatients. Chi-squared testing with odds ratios and 95% confidence intervals determined associations between the effect of control measures studied and MRSA detection rates. RESULTS: New MRSA detection was 21.6 per 1000 admissions before refurbishment compared with 20.4 per 1000 admissions to the surgical ward after refurbishment. New MRSA detection averaged 6.4 per 1000 hospital admissions before the introduction of record flagging and patient cohorting, compared with 6.2 per 1000 admissions after. CONCLUSION: Neither ward refurbishment, nor introduction of flagging, significantly reduced rates of colonization during the study period. In hospitals that receive MRSA-colonized patients and provide intensive care facilities, spread of MRSA is a major problem. Effective containment demands separate wards for MRSA-colonized and non-colonized patients. The need for such containment should be considered in design of the modern hospital.

Phages for methicillin-resistant Staphylococcus aureus: an international trial.

An internationally agreed and validated set of phages is used worldwide for the typing of strains of Staphylococcus aureus of human origin. However, because of the sometimes reduced susceptibility of methicillin-resistant strains (MRSA) to these phages, some of the national typing centres use locally isolated and characterized sets of experimental phages. In this trial, 42 such phages were distributed to 6 centres and tested against 744 isolates of MRSA with the intention of defining a phage set to augment the international set. The use of these experimental phages increased the percentage typability from 75% with the international set to 93% and the number of identifiable lytic patterns from 192 to 424. A subset of 10 experimental phages was selected. When this subset was compared with the experimental panel, the typability rate was 91% and 370 distinct patterns were obtained. This subset of phages has been distributed for international trial.

Evolution of an endemic methicillin-resistant Staphylococcus aureus population in an Australian hospital from 1967 to 1996.

The evolution over 30 years of a population of methicillin-resistant Staphylococcus aureus (MRSA) from a tertiary referral hospital was studied by phylogenetic analysis of SmaI-generated restriction fragment length polymorphisms (RFLPs). The results suggest that a new clone of MRSA appeared at the hospital in the early 1980s, which, although usually retaining its ancestral phage-type, developed four different RFLP pulsotypes in the next 16 years. This finding indicates that multiple RFLP patterns in MRSA do not necessarily represent multiple clones deriving from different mec gene transfer events. Such variation within a clone may be significant in the interpretation of RFLP patterns during outbreaks and emphasizes the need to use two typing methods in studies of such populations. Since the appearance of new clones of MRSA is a relatively rare event, cross-infection control is paramount in the prevention of MRSA dissemination.

Methicillin-resistant Staphylococcus aureus in a cystic fibrosis unit.

Methicillin-resistant Staphylococcus aureus (MRSA) infection in a cystic fibrosis (CF) unit was investigated. Two typing methods, phage-typing and restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE) and phylogenetic analysis, showed that nonsocomial transmission of MRSA from the general hospital population had occurred. One instance of possible transmission between two patients was identified. However, transmission between two family members did not occur indicating a minimal risk of MRSA acquisition from social contact compared with hospital admission. This study supports policies for limiting CF-patient admission to hospital but transmission of MRSA does not appear to be a reason for limiting social contact with other CF patients.

Evaluation of a decision support system for pressure ulcer prevention and management: preliminary findings.

A decision support system for prevention and management of pressure ulcers was developed based on AHCPR guidelines and other sources. The system was implemented for 21 weeks on a 20-bed clinical care unit. Fifteen nurses on that unit volunteered as subjects of the intervention to see whether use of the system would have a positive effect on their knowledge about pressure ulcers and on their decision-making skills related to this topic. A similar care unit was used as a control. In addition, the system was evaluated by experts for its instructional adequacy, and by end users for their satisfaction with the system. Preliminary results show no effect on knowledge about pressure ulcers and no effect on clinical decision making skills. The system was rated positively for instructional adequacy, and positively for user satisfaction. User interviews related to satisfaction supplemented the quantitative findings. A discussion of the issues of conducting experiments like this in today's clinical environment is included.

A knowledge-based decision support system for prevention and treatment of pressure ulcers.

As part of a research project intended to provide problem-based knowledge to clinicians at the point of care, we have developed a system that supports the nurse's development of patient-specific, guideline-based treatment plans for patients who have pressure ulcers or are at risk for developing them. The system captures coded data about assessment, diagnosis and interventions using a point-and-click interface. Knowledge is accessible to the user via: 1) hypertext links from the data entry screens; 2) explicit entry into an indexed version of the guideline; 3) imbedded knowledge-based rules that critique the diagnosis and offer guidance for treatment; and 4) explicit entry into interactive algorithms. The system has been implemented experimentally on one care unit at our hospital, where its impact will be assessed in comparison with a control unit. Data on 113 patients were entered during the 21-week experimental period. The system is being evaluated for its instructional adequacy, its impact on clinicians' decision-making and knowledge, and on processes of care. Users' perceptions of the system are also being evaluated. Dissemination issues in the context of today's health care environment are addressed.

Fine needle aspiration biopsy of the thyroid gland in patients with prior Graves' disease treated with radioactive iodine. Morphologic findings and potential pitfalls.

OBJECTIVE: To evaluate the morphologic findings and their potential pitfalls in fine needle aspiration biopsies (FNAB) of thyroid glands obtained following radioactive iodine (RaI) (131I) treatment for Graves' disease. STUDY DESIGN: Study of thyroid FNAB specimens from six patients with prior Graves' disease treated with RaI who developed palpable nodules and had subsequent thyroid resections. RESULTS: The cytologic changes attributed to radiation were quite variable among the six cases and were so pronounced in one case that a false positive diagnosis of papillary carcinoma was made even though a history of RaI had been provided. The FNAB specimen from the second case, submitted without a history of RaI treatment, was diagnosed as suspicious for papillary carcinoma. The smears from patient 3 were signed out descriptively because the pertinent clinical history had not been provided. The FNAB specimens from the last three patients were correctly interpreted because of the history of RaI therapy provided. All six thyroid surgical specimens showed changes consistent with radiation injury, and none contained evidence of malignancy. CONCLUSION: The study's findings demonstrate that the atypia produced by RaI may be severe, leading to an erroneous diagnosis of malignancy. Provision of the appropriate clinical history of Graves' disease treated with RaI may prevent this pitfall.

Effect of Brugia malayi on the growth and proliferation of endothelial cells in vitro.

Athymic mice (C3H/HeN) parasitized by Brugia malayi develop massively dilated lymphatics. The lymphatic endothelial lining is perturbed, and numerous mononuclear and giant cells are closely apposed to the endothelium. The hyperplastic endothelial cells and low opening pressure of the lymphatics suggest abnormal multiplication of these cells may be important in the dilation. We studied the in vitro growth rate of human umbilical vein endothelial cells cultured with adult worms and microfilariae of B. malayi. The tetrazolium salt reduction assays were used to quantify possible direct mitogenic or inhibitory effects. The growth factor-induced proliferation of endothelial cells was significantly suppressed by 44-51% on day 1, 46-81% on day 3, and 45-79% on day 5 in cultures containing adult female worms, which had greater suppressor activity on endothelial cell proliferation than male worms, microfilariae, or soluble adult worm extract. Culture supernatant containing female worm excretory-secretory products significantly inhibited the growth and multiplication of cells, suggesting that adult female worms release antigens or proteins that have inhibitory activity on growth factors necessary for endothelial cell proliferation in vitro. Excess human recombinant epidermal growth factor and bovine brain extract partly reversed the inhibitory activity of worms in culture and restored the endothelial cell proliferation when incubated with worm culture supernatant. Indomethacin and BW 775Hcl failed to restore normal endothelial proliferation in the presence of female worms, suggesting that parasite-derived prostanoids and cyclooxygenase products did not cause the inhibition. Lymph from dilated lymphatics, but not serum from infected mice, increased the proliferation of cells in vitro. Together, these data demonstrate that excretory-secretory products of B. malayi parasites suppress vascular endothelial proliferation in vitro. Furthermore, increases in the number of these cells in vitro in the presence of lymph suggest that parasite-induced host factors may be important in modulating the degree of proliferation.

Regulatory cytokines in the lymphatic pathology of athymic mice infected with Brugia malayi.

To investigate whether Brugia malayi-induced lymphatic inflammation is due to production of pro-inflammatory cytokines, we determined the lymph and serum levels of interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-alpha and granulocyte-macrophage colony stimulating factor (GM-CSF) using enzyme immunoassays. Serum from normal and infected mice did not show elevated cytokine concentrations. Samples of lymph from parasitized lymphatics had significantly increased levels of IL-1 (range = 6-1620 pg/ml), IL-6 (19-17,800 pg/ml), TNF-alpha (19-2000 pg/ml) and GM-CSF (4-275 pg/ml). The anti-inflammatory cytokine IL-4 (7-12 pg/ml) was in the normal range and no increase in interferon (INF)-gamma was detected in lymph samples. The data suggest that increased levels of mediators or cytokines localized in the lymphatics may be important contributors to massive lymphatic dilation and inflammation.