Variability in Test Interval Is Linked to Glycated Haemoglobin (HbA1c) Trajectory over Time.

Aims: We previously showed that the glycated haemoglobin (HbA1c) testing frequency links to diabetes control. Here, we examine the effect of variability in test interval, adjusted for the frequency, on change in HbA1c (ΔHbA1c). Materials & Methods. HbA1c results were collected on 83,872 people with HbA1c results at baseline and 5 years (±3 months) later and ≥6 tests during this period. We calculated the standard deviation (SD) of test interval for each individual and examined the link between deciles of SD of the test interval and ΔHbA1c level, stratified by baseline HbA1c. Results: In general, less variability in testing frequency (more consistent monitoring) was associated with better diabetes control. This was most evident with moderately raised baseline HbA1c levels (7.0-9.0% (54-75 mmol/mol)). For example, in those with a starting HbA1c of 7.0-7.5% (54-58 mmol/mol), the lowest SD decile was associated with little change in HbA1c over 5 years, while for those with the highest decile, HbA1c rose by 0.4-0.6% (4-6 mmol/mol; p < 0.0001). Multivariate analysis showed that the association was independent of the age/sex/hospital site. Subanalysis suggested that the effect was most pronounced in those aged <65 years with baseline HbA1c of 7.0-7.5% (54-58 mmol/mol). We observed a 6.7-fold variation in the proportion of people in the top-three SD deciles across general practices. Conclusions: These findings indicate that the consistency of testing interval, not the just number of tests/year, is important in maintaining diabetes control, especially in those with moderately raised HbA1c levels. Systems to improve regularity of HbA1c testing are therefore needed, especially given the impact of COVID-19 on diabetes monitoring.

Selecting Core Outcomes for Randomised Effectiveness trials In Type 2 diabetes (SCORE-IT): a patient and healthcare professional consensus on a core outcome set for type 2 diabetes.

Objectives: Heterogeneity in outcomes measured across trials of glucose-lowering interventions for people with type 2 diabetes impacts on the ability to compare findings and may mean that the results have little importance to healthcare professionals and the patients that they care for. The SCORE-IT study (Selecting Core Outcomes for Randomised Effectiveness trials In Type 2 diabetes) has addressed this issue by establishing consensus on the most important outcomes for non-surgical interventions for hyperglycemia in type 2 diabetes. Research design and methods: A comprehensive list of outcomes was developed from registered clinical trials, online patient resources, qualitative literature and long-term studies in the field. This list was then scored in a two-round online Delphi survey completed by healthcare professionals, people with type 2 diabetes, researchers in the field and healthcare policymakers. The results of this online Delphi were discussed and ratified at a face-to-face consensus meeting. Results: 173 people completed both rounds of the online survey (116 people with type 2 diabetes, 37 healthcare professionals, 14 researchers and 6 policymakers), 20 of these attended the consensus meeting (13 people with type 2 diabetes and 7 healthcare professionals). Consensus was reached on 18 core outcomes across five domains, which include outcomes related to diabetes care, quality of life and long-term diabetes-related complications. Conclusions: Implementation of the core outcome set in future trials will ensure that outcomes of importance to all stakeholders are measured and reported, enhancing the relevance of trial findings and facilitating the comparison of results across trials.

The frequency of testing for glycated haemoglobin, HbA1c, is linked to the probability of achieving target levels in patients with suboptimally controlled diabetes mellitus.

Background We previously showed, in patients with diabetes, that >50% of monitoring tests for glycated haemoglobin (HbA1c) are outside recommended intervals and that this is linked to diabetes control. Here, we examined the effect of tests/year on achievement of commonly utilised HbA1c targets and on HbA1c changes over time. Methods Data on 20,690 adults with diabetes with a baseline HbA1c of >53 mmol/mol (7%) were extracted from Clinical Biochemistry Laboratory records at three UK hospitals. We examined the effect of HbA1c tests/year on (i) the probability of achieving targets of ≤53 mmol/mol (7%) and ≤48 mmol/mol (6.5%) in a year using multi-state modelling and (ii) the changes in mean HbA1c using a linear mixed-effects model. Results The probabilities of achieving ≤53 mmol/mol (7%) and ≤48 mmol/mol (6.5%) targets within 1 year were 0.20 (95% confidence interval: 0.19-0.21) and 0.10 (0.09-0.10), respectively. Compared with four tests/year, having one test or more than four tests/year were associated with lower likelihoods of achieving either target; two to three tests/year gave similar likelihoods to four tests/year. Mean HbA1c levels were higher in patients who had one test/year compared to those with four tests/year (mean difference: 2.64 mmol/mol [0.24%], p<0.001). Conclusions We showed that ≥80% of patients with suboptimal control are not achieving commonly recommended HbA1c targets within 1 year, highlighting the major challenge facing healthcare services. We also demonstrated that, although appropriate monitoring frequency is important, testing every 6 months is as effective as quarterly testing, supporting international recommendations. We suggest that the importance HbA1c monitoring frequency is being insufficiently recognised in diabetes management.

Selecting Core Outcomes for Randomised Effectiveness trials In Type 2 Diabetes (SCORE-IT): study protocol for the development of a core outcome set.

BACKGROUND: Type 2 diabetes is characterised by abnormal glucose metabolism, and treatment is aimed at normalising glycaemia. Outcomes measured in clinical trials should be meaningful to patients, health care professionals and researchers, yet there is heterogeneity in the outcomes used across trials of glucose-lowering interventions. This inconsistency affects the ability to compare findings and may mean that the results have little importance to health care professionals and the patients for whom they care. The SCORE-IT study aims to develop a core outcome set (COS) for use in all trials of glucose-lowering interventions for people with type 2 diabetes. METHODS/DESIGN: This study will involve three key stages in the development of a COS: (1) A list of outcomes will be identified from multiple sources, specifically registered clinical trials, online patient resources, the qualitative literature and landmark studies identified by a Study Steering Committee. (2) The list of outcomes will be scored by multiple stakeholder groups in a two-round online international Delphi survey. (3) The results of the online Delphi will be summarised and discussed at a face-to-face consensus meeting with representation from all stakeholder groups. DISCUSSION: The SCORE-IT study aims to develop an internationally relevant set of core outcomes for use in future trials of glucose-lowering interventions for type 2 diabetes. The use of a COS will improve the consistency of outcomes, allowing results of studies to be compared and combined and for new effective treatments to made available more quickly. TRIAL REGISTRATION: The COS study, of which this is a part, is registered in the Core Outcome Measures in Effectiveness Trials (COMET) database, http://www.comet-initiative.org/studies/details/956 . Registered January 2017.

A Reagentless, Screen-Printed Amperometric Biosensor for the Determination of Glutamate in Food and Clinical Applications.

A reagentless biosensor has been successfully developed to measure glutamate in food and clinical samples. The enzyme, glutamate dehydrogenase (GLDH) and the cofactor, nicotinamide adenine dinucleotide (NAD+) are fully integrated onto the surface of a Meldola's Blue screen-printed carbon electrode (MB-SPCE). The biological components are immobilized by utilizing unpurified multi-walled carbon nanotubes (MWCNT's) mixed with the biopolymer chitosan (CHIT), which are drop-coated onto the surface of the MB-SPCE in a layer-by-layer fashion. Meldola's Blue mediator is also incorporated into the biosensor cocktail in order to increase and facilitate electron shuttling between the reaction layers and the surface of the electrode. The loadings of each component are optimized by using amperometry in stirred solution at a low fixed potential of +0.1 V. The optimum temperature and pH are also determined using this technique. Quantification of glutamate in real samples is performed using the method of standard addition. The method of standard addition involves the addition of a sample containing an unknown concentration of glutamate, followed by additions of known concentrations of glutamate to a buffered solution in the cell. The currents generated by each addition are then plotted and the resulting line is extrapolated in order to determine the concentration of glutamate in the sample (Pemberton et al., Biosens Bioelectron 24:1246-1252, 2009). This layer-by-layer approach holds promise as a generic platform for the fabrication of reagentless biosensors.

A novel reagentless glutamate microband biosensor for real-time cell toxicity monitoring.

A reagentless glutamate biosensor was applied to the determination of glutamate released from liver hepatocellular carcinoma cells (HepG2) in response to toxic challenge from various concentrations of paracetamol. A screen printed carbon electrode (SPCE) containing the electrocatalyst Meldola's Blue (MB-SPCE) served as the electron mediator for the oxidation of NADH. A mixture of the enzyme glutamate dehydrogenase (GLDH), cofactor nicotinamide adenine dinucleotide (NAD(+)) and the biopolymer chitosan (CHIT) were drop-coated onto the surface of the transducer (MB-SPCE) in a simple one step fabrication process. The reagentless biosensor was used with amperometry in stirred solution at an applied potential of +0.1 V (vs. Ag/AgCl). All experiments were carried out at the following conditions: pH 7, temperature 37 °C, atmosphere 5% CO2. The linear range of the device was found to be 25-125 µM in phosphate buffer (75 mM, containing 0.05 M NaCl) and 25-150 µM in cell culture medium. The limits of detection (LOD) were found to be 1.2 µM and 4.2 µM based on three times signal to noise, using PBS and culture medium respectively. The sensitivity was calculated to be 106 nA µM(-1) cm(-2) and 210 nA µM(-1) cm(-2) in PBS and cell medium respectively. The response time was ∼60 s in an agitated solution. HepG2 cells were exposed to various concentrations of paracetamol (1 mM, 5 mM and 10 mM) in order to investigate the drug-induced release of glutamate into the culture medium in real time. Two toxicity studies were investigated using different methods of exposure and analysis. The first method consisted of a single measurement of the glutamate concentration, using the method of standard addition, after 24 h incubation. The concentrations of glutamate were found to be 52 µM, 93 µM and 177 µM, released on exposure to 1 mM, 5 mM and 10 mM paracetamol respectively. The second method involved the continuous monitoring of glutamate released from HepG2 cells upon exposure to paracetamol over 8 h. The concentrations of glutamate released in the presence of 1 mM, 5 mM and 10 mM paracetamol, increased in proportion to the drug concentration, ie: 16 µM, 28 µM and 62 µM respectively. This result demonstrates the feasibility of using this approach to monitor early metabolic changes after exposure to a model toxic compound.

Reduced testing frequency for glycated hemoglobin, HbA1c, is associated with deteriorating diabetes control.

OBJECTIVE: We previously showed that in patients with diabetes mellitus, glycated hemoglobin (HbA1c) monitoring outside international guidance on testing frequency is widespread. Here we examined the relationship between testing frequency and diabetes control to test the hypothesis that retest interval is linked to change in HbA1c level. RESEARCH DESIGN AND METHODS: We examined repeat HbA1c tests (400,497 tests in 79,409 patients, 2008-2011) processed by three U.K. clinical laboratories. We examined the relationship between retest interval and 1) percentage change in HbA1c and 2) proportion of cases showing a significant HbA1c rise. The effect of demographics factors on these findings was also explored. RESULTS: Our data showed that the optimal testing frequency required to maximize the downward trajectory in HbA1c was four times per year, particularly in those with an initial HbA1c of ≥7% (≥53 mmol/mol), supporting international guidance. Testing 3-monthly was associated with a 3.8% reduction in HbA1c compared with a 1.5% increase observed with annual testing; testing more frequently provided no additional benefit. Compared with annual monitoring, 3-monthly testing was associated with a halving of the proportion showing a significant rise in HbA1c (7-10 vs. 15-20%). CONCLUSIONS: These findings provide, in a large, multicenter data set, objective evidence that testing outside guidance on HbA1c monitoring frequency is associated with a significant detrimental effect on diabetes control. To achieve the optimum downward trajectory in HbA1c, monitoring frequency should be quarterly, particularly in cases with suboptimal HbA1c. While this impact appears small, optimizing monitoring frequency across the diabetes population may have major implications for diabetes control and comorbidity risk.

Microfabricated glucose biosensor for culture well operation.

A water-based carbon screen-printing ink formulation, containing the redox mediator cobalt phthalocyanine (CoPC) and the enzyme glucose oxidase (GOx), was investigated for its suitability to fabricate glucose microbiosensors in a 96-well microplate format: (1) the biosensor ink was dip-coated onto a platinum (Pt) wire electrode, leading to satisfactory amperometric performance; (2) the ink was deposited onto the surface of a series of Pt microelectrodes (10-500 µm diameter) fabricated on a silicon substrate using MEMS (microelectromechanical systems) microfabrication techniques: capillary deposition proved to be successful; a Pt microdisc electrode of ≥100 µm was required for optimum biosensor performance; (3) MEMS processing was used to fabricate suitably sized metal (Pt) tracks and pads onto a silicon 96 well format base chip, and the glucose biosensor ink was screen-printed onto these pads to create glucose microbiosensors. When formed into microwells, using a 340 µl volume of buffer, the microbiosensors produced steady-state amperometric responses which showed linearity up to 5 mM glucose (CV=6% for n=5 biosensors). When coated, using an optimised protocol, with collagen in order to aid cell adhesion, the biosensors continued to show satisfactory performance in culture medium (linear range to 2 mM, dynamic range to 7 mM, CV=5.7% for n=4 biosensors). Finally, the operation of these collagen-coated microbiosensors, in 5-well 96-well format microwells, was tested using a 5-channel multipotentiostat. A relationship between amperometric response due to glucose, and cell number in the microwells, was observed. These results indicate that microphotolithography and screen-printing techniques can be combined successfully to produce microbiosensors capable of monitoring glucose metabolism in 96 well format cell cultures. The potential application areas for these microbiosensors are discussed.

Development of a sandwich format, amperometric screen-printed uric acid biosensor for urine analysis.

A screen-printed carbon electrode (SPCE) incorporating the electrocatalyst cobalt phthalocyanine (CoPC), fabricated using a water-based ink formulation, has been investigated as the base transducer for a uric acid biosensor. A sandwich biosensor was fabricated by first depositing cellulose acetate (CA) onto this transducer (CoPC-SPCE), followed by uricase (UOX) and finally a polycarbonate (PC) membrane; this device is designated PC-UOX-CA-CoPC-SPCE. This biosensor was used in conjunction with chronoamperometry to optimize the conditions for the analysis of urine: temperature, 35°C; buffer, pH 9.2; ionic strength, 50 mM; uricase, 0.6 U; incubation time, 180 s. The proposed biosensor was applied to urine from a healthy subject. The precision determined on unspiked urine (n=6) was 5.82%. Urine was fortified with 0.225 mM UA, and the resulting precision and recovery were 4.21 and 97.3%, respectively. The linear working range of the biosensor was found to be 0.015 to 0.25 mM (the former represents the detection limit), and the sensitivity was calculated to be 2.10 µA/mM.

Complications and reintervention following elective open abdominal aortic aneurysm repair: a 10-year retrospective analysis.

INTRODUCTION: Recent data have shown higher rates of graft related complication or reintervention in patients undergoing endovascular aneurysm repair compared with open aneurysm surgery (OAS). However, there are fewer data available regarding procedure related reinterventions following OAS. The aim of this study was to investigate the incidence of procedure related complications and reintervention following elective open abdominal aortic aneurysm repair. METHODS: This was a retrospective analysis of prospectively collected data from the dedicated Portsmouth POSSUM database. Data from 361 patients (median age: 72 years, 91.4% male) who underwent elective OAS between 1993 and 2004 were analysed. The incidences of early and late complications and subsequent reintervention were investigated. RESULTS: The median follow-up duration was 10 years 4 months (range: 5 years - 16 years 4 months). There were 52 reinterventions in the follow-up period. Of these, 34.6% were for incisional hernias or small bowel obstruction with the majority of the remaining laparotomies performed for bleeding or distal ischaemic complications. Almost two-thirds (63.5%) of reinterventions occurred in the first 30 days. There were 30 emergency readmissions to the acute surgical wards that did not require reintervention. CONCLUSIONS: OAS carries a significant reintervention rate. In this study, 54% of reinterventions were directly related to laparotomy.

Inappropriate requesting of glycated hemoglobin (Hb A1c) is widespread: assessment of prevalence, impact of national guidance, and practice-to-practice variability.

BACKGROUND: Estimates suggest that approximately 25% of requests for pathology tests are unnecessary. Even in diabetes, for which international guidance provides recommended testing frequency, considerable variability in requesting practice exists. Using the diabetes marker, Hb A(1c), we examined (a) the prevalence of under- and overrequesting, (b) the impact of international guidance on prevalence, and (c) practice-to-practice variability. METHODS: We examined Hb A(1c) requests (519 664 requests from 115 730 patients, January 2001 to March 2011) processed by the Clinical Biochemistry Department, University Hospital of North Staffordshire, and prevalence of requesting outside guidance from intervals between requests was calculated. Requests were classified as "appropriate," "too soon," or "too late." We also assessed the effect of demographic factors and publication of guidance, along with between-practice variability, on prevalence. RESULTS: Only 49% of requests conformed to guidance; 21% were too soon and 30% were too late. Underrequesting was more common in primary care, in female patients, in younger patients, and in patients with generally poorer control (all P < 0.001); the reverse generally was true for overrequesting. Publication of guidance (e.g., American Diabetes Association, UK National Institute for Health and Clinical Excellence) had no significant impact on under- or overrequesting rates. Prevalence of inappropriate requests varied approximately 6-fold between general practices. CONCLUSIONS: Although overrequesting was common, underrequesting was more prevalent, potentially affecting longer-term health outcomes. National guidance appears to be an ineffective approach to changing request behavior, supporting the need for a multisystem approach to reducing variability.

A screen-printed microband glucose biosensor system for real-time monitoring of toxicity in cell culture.

Microband biosensors, screen-printed from a water-based carbon ink containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, were used to monitor glucose levels continuously in buffer and culture medium. Five biosensors were operated amperometrically (E(app) of +0.4V), in a 12-well tissue culture plate system at 37°C, using a multipotentiostat. After 24 h, a linear calibration plot was obtained from steady-state current responses for glucose concentrations up to 10 mM (dynamic range 30 mM). Within the linear region, a correlation coefficient (R(2)) of 0.981 was obtained between biosensor and spectrophotometric assays. Over 24 h, an estimated 0.15% (89 nmol) of the starting glucose concentration (24 mM) was consumed by the microbiosensor. The sensitivity of the biosensor response in full culture medium was stable between pHs 7.3 and 8.4. Amperometric responses for HepG2 monolayer cultures decreased with time in inverse proportionality to cell number (for 0 to 10(6) cell/ml), as glucose was being metabolised. HepG2 3D cultures (spheroids) were also shown to metabolise glucose, at a rate which was independent of spheroid age (between 6 and 15 days). Spheroids were used to assay the effect of a typical hepatotoxin, paracetamol. At 1 mM paracetamol, glucose uptake was inhibited by 95% after 6 h in culture; at 500 µM, around 15% inhibition was observed after 16 h. This microband biosensor culture system could form the basis for an in vitro toxicity testing system.

Fabrication of microband glucose biosensors using a screen-printing water-based carbon ink and their application in serum analysis.

Microband glucose biosensors were fabricated by screen-printing a water-based carbon ink formulation containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, then insulating and sectioning through the thick (20mum) film to expose a 3mm-long working electrode edge. The performance of these biosensors for glucose analysis was investigated at 25 degrees C. Voltammetry in glucose-containing buffer solutions established that an operating potential of +0.4V vs. Ag/AgCl was suitable for analysis under both stirring and quiescent conditions. The influence of pH on biosensor performance was established and an operational pH of 8.0 was selected. Steady-state responses were obtained under quiescent conditions, suggesting a mixed mechanism predominated by radial diffusion, indicative of microelectrode behaviour. Calibration studies obtained with these biosensors showed steady-state currents that were linearly dependent on glucose concentration from the limit of detection (0.27mM) up to 2.0mM, with a precision for replicate biosensors of 6.2-10.7%. When applied to the determination of glucose in human serum, the concentration compared favourably to that determined by a spectroscopic method. These results have demonstrated a simple means of fabricating biosensors for glucose measurement and determination in situations where low-current real-time monitoring under quiescent conditions would be desirable.

A microband lactate biosensor fabricated using a water-based screen-printed carbon ink.

The present study demonstrated for the first time that screen-printed carbon microband electrodes fabricated from water-based ink can readily detect H(2)O(2) and that the same ink, with the addition of lactate oxidase, can be used to construct microband biosensors to measure lactate. These microband devices were fabricated by a simple cutting procedure using conventional sized screen-printed carbon electrodes (SPCEs) containing the electrocatalyst cobalt phthalocyanine (CoPC). These devices were characterised with H(2)O(2) using several electrochemical techniques. Cyclic voltammograms were found to be sigmoidal; a current density value of 4.2 mA cm(-2) was obtained. A scan rate study revealed that the mass transport mechanism was a mixture of radial and planar diffusion. However, a further amperometric study under quiescent and hydrodynamic conditions indicated that radial diffusion predominated. A chronoamperometric study indicated that steady-state currents were obtained with these devices for a variety of H(2)O(2) concentrations and that the currents were proportional to the analyte concentration. Lactate microband biosensors were then fabricated by incorporating lactate oxidase into the water-based formulation prior to printing and then cutting as described. Voltammograms demonstrated that lactate oxidase did not compromise the integrity of the electrode for H(2)O(2) detection. A potential of +400 mV was selected for a calibration study, which showed that lactate could be measured over a dynamic range of 1-10mM which was linear up to 6mM; a calculated lower limit of detection of 289 microM was ascertained. This study provides a platform for monitoring cell metabolism in-vitro by measuring lactate electrochemically via a microband biosensor.

Application of screen-printed microband biosensors to end-point measurements of glucose and cell numbers in HepG2 cell culture.

Microband glucose biosensors were produced by insulating and sectioning through a screen-printed, water-based carbon electrode containing cobalt phthalocyanine redox mediator and glucose oxidase enzyme. Under quiescent conditions at 37 degrees C, at an operating potential of +0.4V, they produced an amperometric response to glucose in buffer solutions with a sensitivity of 26.4 nA/mM and a linear range of 0.45 to 9.0 mM. An optimal pH value of 8.5 was obtained under these conditions, and a value for activation energy of 40.55 kJ mol(-1) was calculated. In culture medium (pH 7.3), a sensitivity of 13 nA/mM was obtained and the response was linear up to 5 mM with a detection limit of 0.5 mM. The working concentration was up to 20 mM glucose with a precision of 11.3% for replicate biosensors (n=4). The microband biosensors were applied to determine end-point glucose concentrations in culture medium by monitoring steady-state current responses 400 s after transfer of the biosensors into different sample solutions. In conjunction with cultures of HepG2 (human Caucasian hepatocyte carcinoma) cells, current responses obtained in 24-h supernatants showed an inverse correlation (R(2)=0.98) with cell number, indicating that the biosensors were applicable for monitoring glucose metabolism by cells and of quantifying cell number. Glucose concentrations determined using the biosensor assay were in good agreement, for concentrations up to 20mM, with those determined spectrophotometrically (R(2)=0.99). This method of end-point glucose determination was used to provide an estimated rate of glucose uptake for HepG2 cells of 7.9 nmol/(10(6) cells min) based on a 24-h period in culture.

Development of a screen-printed carbon electrochemical immunosensor for picomolar concentrations of estradiol in human serum extracts.

Investigations into the development of a prototype electrochemical immunosensor for estradiol (E(2)) are described. After optimising reagent loadings in a 96-well enzyme-linked immunosorbent assay (ELISA), antibodies (rabbit anti-mouse IgG and monoclonal mouse anti-E(2)) were immobilised by passive adsorption onto the surface of screen-printed carbon electrodes (SPCEs). A competitive immunoassay was then performed using an alkaline-phosphatase (ALP)-labelled E(2) conjugate. Calibration plots for E(2) buffer standards, performed colorimetrically on the SPCEs using a para-nitrophenyl phosphate substrate solution, were in good agreement with ELISA calibration plots. Electrochemical measurements were then performed using differential pulse voltammetry (DPV) following the production of 1-naphthol from 1-naphthyl phosphate. The calibration plot of DPV peak current versus E(2) concentration showed a measurable range of 25-500 pg/ml with a detection limit of 50 pg/ml. A coefficient of variation of between 13.0 and 15.6% was obtained for repeat measurements. The immunosensor was applied to the determination of E(2) in spiked serum, following an extraction step with diethyl ether. A mean recovery for the method of 102.5% was obtained with a CV of 19.1%. The options available for further development of the sensor regarding precision, limit of detection and direct sample analysis are discussed.

First Report of Infection of Lygodium microphyllum by Puccinia lygodii, a Potential Biocontrol Agent of an Invasive Fern in Florida.

Lygodium microphyllum (Cav.) R.Br. (Old World climbing fern), in the family Schizaeaceae, is one of the most invasive (Category I in Florida) weeds in Florida. It has invaded more than 50,000 ha of wetlands and moist habitats in southern Florida and is rapidly spreading in new areas of the Everglades (3). The search and evaluation of biocontrol agents for this fern is currently in progress. Puccinia lygodii (Har.) Arth. (Uredinales) (1), previously recorded on L. volubile Sw. and L. venustum Sw. in South America (2), attacks foliage and severely damages L. japonicum Thunb. (Japanese climbing fern) vines in northern and central Florida (4). We hypothesized that since L. japonicum occurred mainly in northern and central Florida, P. lygodii did not have opportunity to interact with L. microphyllum, which primarily occurs in southern Florida. Therefore, we used two inoculation methods to test the possible pathogenicity of P. lygodii on the new host, L. microphyllum. Method-I was designed to imitate a seminatural inoculation technique in which three containerized (0.45-L capacity) L. microphyllum test plants (15- to 30-cm-high sporelings) were intermixed among a group of containerized (5.0-L capacity) P. lygodii-infected L. japonicum plants (source of inoculum) in a glasshouse. In Method-II, uredospores obtained from pustules on diseased L. japonicum foliage were adjusted to 1 × 106 uredospores/ml and then misted on three L. microphyllum sporelings (same size as in Method-I) until foliage was completely wet. The plants were then covered individually with a plastic bag for 3 days to facilitate spore germination and infection. In both methods, three L. japonicum sporelings of similar size as L. microphyllum were intermixed among diseased L. japonicum plants as a positive control. All test and infected plants were placed on 6-cm-high trays filled two-thirds with water and exposed to diffused daylight and a temperature range of 20 to 35°C in a glasshouse. These plants were monitored for the development of rust symptoms (halos and rust pustules) development for 8 weeks. Minute cinnamon flakes that developed into eruptive pustules were seen on the lower surface of the pinnules approximately 42 and 28 days after treatment initiation (in both methods) for L. microphyllum and L. japonicum (positive control), respectively. Each method was repeated twice. Dimensions (29.7 [±3.7] × 23.5 [±2.6] µm) and morphology of urediniospores from pustules on inoculated L. microphyllum were similar to those reported for P. lygodii on other host systems (1,2,4). To our knowledge, this is the first report demonstrating the infection of P. lygodii on L. microphyllum. The potential use of P. lygodii as a classical bio-control agent of L. microphyllum in southern Florida will be further investigated. References: (1) J. C. Arthur. Bull. Torrey Bot. Club 51:55, 1924. (2) J. W. McCain et al. Mycotaxon 39:281, 1990. (3) R. W. Pemberton. SIDA 20:1759, 2003. (4) M. B. Rayachhetry et al. Plant Dis. 85:232, 2000.

Nontarget effects--the Achilles' heel of biological control? Retrospective analyses to reduce risk associated with biocontrol introductions.

Controversy exists over ecological risks in classical biological control. We reviewed 10 projects with quantitative data on nontarget effects. Ten patterns emerged: (a) Relatives of the pest are most likely to be attacked; (b) host-specificity testing defines physiological host range, but not ecological range; (c) prediction of ecological consequences requires population data; (d) level of impact varied, often in relation to environmental conditions; (e) information on magnitude of nontarget impact is sparse; (f) attack on rare native species can accelerate their decline; (g) nontarget effects can be indirect; (h) agents disperse from agroecosystems; (i) whole assemblages of species can be perturbed; and (j) no evidence on adaptation is available in these cases. The review leads to six recommendations: Avoid using generalists or adventive species; expand host-specificity testing; incorporate more ecological information; consider ecological risk in target selection; prioritize agents; and pursue genetic data on adaptation. We conclude that retrospective analyses suggest clear ways to further increase future safety of biocontrol.